IL285032A - De-immunized, shiga toxin a subunit scaffolds and cell-targeting molecules comprising the same - Google Patents

De-immunized, shiga toxin a subunit scaffolds and cell-targeting molecules comprising the same

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IL285032A
IL285032A IL285032A IL28503217A IL285032A IL 285032 A IL285032 A IL 285032A IL 285032 A IL285032 A IL 285032A IL 28503217 A IL28503217 A IL 28503217A IL 285032 A IL285032 A IL 285032A
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cell
shiga toxin
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IL285032A
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Molecular Templates Inc
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    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Description

WO 2016/196344 PCT/US2016/034778 1 DE-IMMUNIZED, SHIGA TOXIN A SUBUNIT SCAFFOLDS AND CELL- TARGETING MOLECULES COMPRISING THE SAME TECHNICAL FIELD [1] The present invention relate to sShiga toxin effector polypeptides derived, from tiie A Subunit sof natural occlyurr ingShiga toxins, that comprise a combination of mutations providing (1) de-immunization, (2) a reduction in protease sensitivity, and/o (3)r an embedded, T-cell epitope(s wherein); the Shiga toxin effector polypeptides retain one or more Shiga toxin functions, such as, e.g., potent cytotoxicity. In certa embodimein nts, the Shiga toxin effector polypepti desof the present invention (1) exhibit reduced immunogeni potentic inal mammal and/os (2)r are eac hcapabl ofe deliveri ang CD8+ T-cell epitope to the MHC clas I ssystem of a cell in which the polypeptide is present. [2] The present invention also rela testo cell-target molecing ules which comprise a Shiga toxin effector polypeptide of the present invention. The Shiga toxin effector polypepti desof the present invention have uses as scaffolds or components of ceil- targeting molecules, such as, e.g., immunotoxins and ligand-toxin fusions, for killing cells and/or subceilular delivery' of cargos to certai subceln lular compartme suchnts, as, e.g., the delivery' of an embedded, T-ceil epitope to the cytos ol.In general, the Shiga toxin effector polypepti desand cell-targeti molecng ules of the present invention are useful for administrat to ionmulticellula organismsr such, as, e.g., when it is desirabl to e(1) eliminate or reduce non-specifi toxicitiesc (2) ,eliminate or reduce certain immune responses and/or, (3) targe a benet ficia immunel response(s ) to a specif icepitope deliver toed a specifi cell-c type, such as, e.g., the recruitme ofnt CD8+ T-cell s.Hie cell-targeti molecng ules of the prese ntinvention are useful (1) for selective killingly specif iccell type(s amongs) othet celr lsand (2) as therapeutic molecules for treating a variet ofy diseases, disorders and, conditions, including cancers, tumors othe, growr thabnormalit immuneies, disorders and, microbial infections.
BACKGROUND [3] The "magic bullet" conce ispt that therapeutic mays be discovere thatd specifica attally onlyck disease cellsd or pathogens withi an human patient while leaving the patient unharmed. Immunotoxi ligans, nd-to hybrids,xin immuno-WO 2016/196344 PCT/US2016/034778 2 RNases, and other molecular targely teddrugs are descendants of Dr. Paul Ehrlich’s "magic bullet" concept of the early 20th century (Strebha K,rdt Ullrich A, Nat Rev Cancer 8: 473-80 (2008)). The toxin produc edby 5. dysenteriae was named "Shiga toxin" after Dr. Ehrlic’s hassocia Dr.te Kiyoshi Shiga for his discovery7 of this bacterium in 1897. Recently, Shiga toxins have become apprecia tedfor having unique characteristics favorable for use in cell-internali moleculezing fors targete d therapies (see e.g. US20150259428). Shiga toxins may' be combine witd h immunoglobulin domains, ligands and, othe tarr geti moietieng tos create ceil- targe tedtherapeutics (e.g., immunotoxins and ligand-toxi fusions)n that are "magic bullet" s. [4] Shiga toxins, may have advantag eousproperti fores use in therapeut sucicsh , as, e.g., a potent toxi mechann ism effective towar eukard yotic cells abil, ity to drive cellular internaliza andtion, ability to direct subceliula routinr g.Shiga toxins have been synthetica engineerlly fored medical applications by rational alterations to the toxin’s structur chae, racter istandics biological, activitie (see,s e.g. WO 1999/040185, WO 2005/092917, EP1051482, DE69835695, WO 2007/033497, US2009/0156417, JP4339511, US7713915, EP1727827, DE602004027168, EP1945660, JP4934761, EP2228383, US2013/0196928, WO 2014/164680, WO 2014/164693, US20150259428, WO 2015/138435, WO 2015/138452, WO 2015/113005, WO 2015/113007, and WO 2015/191764, eac hof which is incorpora byted, reference herein in its entirety) Shiga. toxin A. Subunit sare stable , enzymatically active and, cytotoxic even if truncat ored fuse dto othe protr ein domains. [5] Major limitations to therapeutic applications involving synthetica lly engineered molecules derive fromd bacterial toxins include both detrimental immunogeni responsesc in recipients and non-specif toxiic cit causeies byd toxic components. Unwante immunod genicit of ay therapeuti productc could result in unfavora conseble quences such, as a reduc edefficacy, the production of neutraliz ing antibodies alter, pharmaced okinet generaics, immunel and hypersensitiv ity reactions, anaphylaxis anaphylactoid, reactions, and constraint on thes number of repeat doses a recipient can safely receive. Reducing the non-specif toxicitic ofy a therapeutic molecule can improve its safety characteris whentics administer to eda. recipient as well as alter its potentia therapeutil windowc by increasing the maximum dosage which can be administer safedely. Because both unwanted WO 2016/196344 PCT/US2016/034778 3 immune response ands non-specifi toxicitiesc can pose significant safety and/or efficacy issue(s) for a drug therapy, reducin org minimizing the probabilities of both is often desirable when developing therapeutic molecules. [6] The stabilit ofy a therape uticor diagnosti moleculc overe time and in specific environme (e.g.nts the human circulat7 orysystem) are importa feant ture ands can affect for which application a molecs ule may be practical employely d.For certa in immunotox orins ligand-toxin fusions, the stabilit ofy the linkage between the toxin and othe componentsr can affect the amount of non-specif toxicityic caused by the release of untargete toxind over time withi then body of a multicellul orgaar nism, !־bus, for molecules comprisin toxing components, certa non-specifiin toxicitiesc are directly related to the stabili ofty the connection between the toxin component and another component, such as, e.g., a cell-targeti component.ng [7] Shiga toxins can be combine withd heterologous epitopes to creat cell-e targe tedtherapeutic whichs deliver chosen epitope cargos for the purpose of inducing desirable immune response (sees WO 2015/113007). These immune response mays be harnes sedby7 therapeu molectic ules for the targete killd ing of specifi celc l-ty withinpes a patient as wel asl to sensitize the patien’s timmune system to identifyin certg ain cells as foreign (i.e. breaking immunotolerance) For . example, the Major Histo-Compatibility (MHC) class I presentati pathwayon may be exploite byd such approac hesto induc thee recruitme ofnt immune cells to tumor loci within a patient and to enhance the recognition of certai neoplasn cellstic by7 immune surveillanc mechanie sms. [8] It would be desirable to have Shiga toxin A Subunit-deriv polyped epti des wit hlow antigenicit lowy, immunogenici and/oty, comprisingr heterologous epitopes but, which reta ain significant level of a Shiga toxin function(s), such as, e.g., potent cytotoxicity, the ability to force cellular internaliza and/otion, ther ability to efficiently route to a desired intracellu location(s)lar Furthermore,. it would be desira bleto have therapeut and/oric diagnos moleculetic comprs isin Shigag toxin A Subunit derived compone ntshaving low antigenici lowty,7 immunogenici highty, stabilit lowy, non-specifi toxicity,c and/or the ability to deliv peptide-er epitope cargos for presentati byon the MHC cla ssI system of a target cel l.In particular, it would be desirable to have cel I-targeting molecule comprisings Shiga toxin A Subunit derive componed ntsthat mainta potentin cytotoxi whilecity 1) reducing the potenti foral unwanted antigenic itiand/ores immunogenicities 2) reducing, the WO 2016/196344 PCT/US2016/034778 4 potenti foral non-specifi toxicitc ies,and/o 3)r having the ability to delive peptide-r epitope cargos for presentati byon tire MHC clas I ssystem of a targ cellet .
SUMMARY OF THE INVENTION [9] The Shiga toxin A Subuni tderive scad ffolds of the present invention eac h comprise a combinat ionof feature (e.g.,s de-immunize sub-region(sd heter), ologous epitope comprisin sub-g region(s a protea), se-cleava resistge antsub-region, and/o a r carboxy-term endopinal, las micreticulum retention/retr signalieva motif)l which make them more useful, such as, e.g., as components of cell-target moleculeing likes immunotoxins and ligand-toxin fusions. Certai combinationn Shiga toxin effector polypepti desof the prese ntinvention are more useful because they provide sever al Shiga toxin effector functions in a single polypepti de,such as, e.g., promoting cellular internalizati direon,ctin sub-cellulag routingr to the cytos ol,ribosome inactivation, and/o deliveringr heterolog T-ceous,ll epitopes to the MHC I cla ss pathway of a cell Certa. cell-in target moleculesing of the prese ntinvention are more useful becaus theye provide a combination of several propert inies a single molecule , such as, e.g., efficient cellular internalizati potenton, cell-target cytotoxicity,ed selecti cytotve oxici de-ity,mmunization, low non-specifi toxicc ityat high dosage s, high stability, CD+ T-cell hyper-immunizat and/orion, the ability to delive a r heterologous T-ce,ll epitope (s)to tire MHC I class pathw ayof a targe cellt . . . . id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10"
[10] Differe embodiment nts of the Shiga toxin effector polypepti desand cell - targeting molecules of the prese ntinvention are described below with refere nceto sets of embodiments numbere #1-11.d Embodiment Set #1 - De-immunize d.Shiga Toxin Effector Polypeptide Comprising an Embedded or Inserted, Heterologous, T-Cell Epitope 11. 11. 11. id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11"
[11] The present invention provides a de-immunized, Shiga toxin effector polypeptide comprising at leas onet insert ored embedded, heterologous epitope (a) and at leas onet disrupted, endogenous, B-cell and/or CD4+ T-cell epitope regio n (b), wherein the heterologous epitope does not overlap with at lea stone disrupted, endogenous, B-cell and/o CD4+r T-cell epitope region; and wherein the Shiga toxin effector polypeptide is capabl ofe exhibiting at leas onet Shiga toxin effector function (see e.g. Figure 1, depicting illustrat examive ples of thre exemplare, y embodiments of the de-immuniz edShiga toxin effector polypept ideof tins WO 2016/196344 PCT/US2016/034778 embodiment set #1 labeled Shiga toxin effecto 1, 2,r and 3). In certa furthein r embodiments, the heterologous epitope is a CD8+ T-cell epitope capable of being presente byd a MHC clas I smolecule of a cell In. certa furthein embodimer nts, the heterologous epitope in (a) is embedded and replace an sequivalen numbert of amino acid residues in a wild-type Shiga toxin polypeptide region such that the Shiga toxin effector polypept idehas the same tota numberl of amino acid residues as does the wild-type Shiga toxin polypeptide region from which it is derive d.In certain furthe r embodiments of any of the above, the de-immunize Shigad, toxin effector polypeptide is capable of exhibiting at leas onet Shiga toxin effector function selected from: directing intracellular routing to a cytosol of a cell in whic theh polypept ideis present, inhibiting a. ribosome function, enzymatically inactiva tinga ribosome, and cytotoxicity. 12. 12. 12. id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12"
[12] In certa embodimein nts, the de-immunized, Shiga toxin effector polypept ide of the prese ntinvention compris (i)es an embedded or inserte heted, rologous T-ce,ll epitope and (ii) a disruption of at leas one,t endogenous B-ce, ll and/or T-cell epitope which does not overlap with the embedded or inserte heted, rolog T-celous, epitopel .
In certai furthen embodimer nts, the Shiga toxin effecto polypeptider is capabl ofe exhibiting at leas onet Shiga toxin effector function, such as, e.g., directi ng intracellula routingr to the endoplasmic reticul umand/o cytosr ofol a ceil in which the polypeptide is present, inhibiting a ribosome function, enzymatica inacllytivating a ribosome, causing cytostas and/oris, causing cytotoxici Inty. certain furthe r embodiments, the heterologous T-ce,ll epitope is a CD8+ T-cell epitope, such as, e.g., with rega rdto a huma nimmune syste m.In certa furtherin embodiments, the heterologous T-ce,ll epitope is capabl ofe being presente byd a MHC clas I s molecule of a cell In. certain further embodiments, the endogenous, T-cell epitope is a CD4+ T-cell epitope, such as, e.g., with rega rdto a human immune system. 13. 13. 13. id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13"
[13] In certa embodimein nts, the de-immunized, Shiga toxin effector polypept ide of the prese ntinvention compris (i)es an embedded or inserte heted, rologous T-ce,ll epitope and (ii) a disruption of at leas one,t endogenous B-ce, ll and/or T-cell epitope regio whichn does not overlap with the embedded or inserte heted, rologous T-cel, l epitope. In certai furthen embodr iment thes, Shiga toxin effector polypeptide is capable of exhibiting at leas onet Shiga toxin effector function, such as, e.g., directing intracel lularoutingr to the endoplasmic reticulum and/o cytosolr of a cell m which the polypept ideis present, inhibiting a ribosome function, enzymatica llyWO 2016/196344 PCT/US2016/034778 6 inactivati a ribosome,ng causing cytostas and/ois, causingr cytotoxicity. In certa in furthe embodimer nts, the heterologous T-ce,ll epitope is a CD8+ T-cell epitope , such as, e.g., wit hregard to a huma nimmune system. In certa furthein r embodiments, the heterologous T-ce,ll epitope is capable of being presente byd a MHC class I molecule of a cell In. certa furthein embodimer nts, the endogenou T-s, cell epitope region is a CD4+ T-cell epitope region, such as, e.g., with rega rdto a human immune system. 14. 14. 14. id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14"
[14] In certa embodimein nts, the de-immunized, Shiga toxin effector polypept ide of tire prese ntinvention compris (i)es an embedde ord inserte heted, rologous T-ce,ll epitope and (ii) a disrupti onof at leas one,t endogenous B-cell, and/or T-cell epitope which does not overlap with the embedded or inserte heted, rolog T-ceous,ll epitope.
In certai furthen embodimer nts, the Shiga toxin effecto polypeptider is capabl ofe exhibiting at lea stone Shiga toxin effector function, such as, e.g., directi ng intracellula routingr to the endoplasm reticiculum and/o cyr tosol of a cell in which the polypeptide is present, inhibiting a. ribosome function, enzymatically inactivating a ribosome, causing cytostas and/oris, causing cytotoxici Inty. certain further embodiments, the heterologous T-ce,ll epitope is a CD8+ T-cell epitope, such as, e.g., with regar tod a. human immune syste m.In certa furtherin embodiments, the heterologous T-ce,ll epitope is capabl ofe being presented by a MHC clas I s molecule of a cell In. certain further embodiments, the endogenous, T-cell epitope is a CD4+ T-cell epitope, such as, e.g., with rega rdto a huma nimmune system. . . . id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15"
[15] In certa embodimein nts, the de-immunized, Shiga toxin effector polypept ide of the prese ntinvention compris (i)es an embedde ord inserte heted, rologous T-ce,ll epitope and (ii) a disrupti onof at least one, endogenous B-ce, ll and/or T-cell epitope region which does not overlap wit hthe embedded or inserte heted, rologous T-ce,ll epitope. In certai furthern embodiment thes, Shiga toxin effector polypeptide is capable of exhibiting at leas onet Shiga toxin effector function, such as, e.g., directing intrac lularel routing to the endoplasmic reticulum and/o cytosolr of a cell in which the polypept ideis present, inhibiting a ribosome function, enzymatic ally inactivati a ribosome,ng causing cytostas and/ois, causingr cytotoxicity. In certa in furthe embodimer nts, the heterologous T-ce,ll epitope is a CD8+ T-cell epitope , such as, e.g., wit hregard to a huma nimmune system. In certa furthein r embodiments, the heterologous T-ce,ll epitope is capable of being presented by a MHC class I molecule of a cell In. certa furthein embodimer nts, the endogenou T-s, WO 2016/196344 PCT/US2016/034778 7 cell epitope regio isn a CD4+ T-cell epitope region, such as, e.g., wit hrega rdto a human immune system. 16. 16. 16. id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16"
[16] In certain em bodiments of Embodiment Set #1, the Shiga toxin effector polypeptide comprises a mutation, relat iveto a. wild-type Shiga toxin A Subunit, in the B-cel and/orl T-cell epitope region selected from the group of natively positioned Shiga toxin A Subunit regions consist ingof: 1-15 of SEQ ID NO: 1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3: 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53 66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3: 141-153 of SEQ ID NO: I or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2, and 210-218 of SEQ IDN0:3; 240-260 of SEQ IDN0:3; 243-257 of SEQ IDNO:1 or SEQ ID NO:2; 254-268 of SEQ ID NO: 1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; 285-293 of SEQ ID NO:1 or SEQ ID NO:2; 4-33 of SEQ ID NO: 1 or SEQ ID NO:2; 34-78 of SEQ ID NO: I or SEQ ID NO:2; 77- 103 of SEQ ID NO: 1 or SEQ ID NO:2; 128-168 of SEQ ID NO: 1 or SEQ ID NO:2; 160-183 of SEQ ID NO: I or SEQ ID NO:2; 236-258 of SEQ ID NO: 1 or SEQ ID NO:2; and 274-293 of SEQ ID NO: 1 or SEQ ID NO:2; or the equivale regnt ion in a Shiga toxin A Subunit or derivative thereof. In certa furthein embodimer nts, there is no disruption which is a carboxy-termi truncanal tion of amino acid residues that overlap with part or all of at leas onet disrupted, endogenous, B-cell and/o T-cellr epitope and/or epitope region. 17. 17. 17. id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17"
[17] In certain embodiments of Embodiment Set «1, tire Shiga toxin effector polypeptide comprises a mutation, relat iveto a. w'ild-type Shiga toxin A Subunit, in the B-cel immunogenic,l amino acid residue selected from the group of natively' positioned Shiga toxin A Subunit amino acid residues: L49, D197, D198, R204, and R205. 18. 18. 18. id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18"
[18] In certain embodimen ofts Embodiment Set # 1, the embedded or inserte d, heterologous T-ce,ll epitope disrupts the endogenous, B-cell and/o T-cellr epitope region is selected from the group of natively positioned Shiga toxin A Subunit regio nsconsisting of: (i) 1-15 of SEQ ID NO: 1 or SEQ ID NO:2; 3—14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO: I or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3: and 53-66 of SEQ IDWO 2016/196344 PCT/US2016/034778 8 NO: 1, SEQ ID NO:2, or SEQ ID NO:3; or the equivalent region in a Shiga toxin A Subunit or derivati thereve of,wherein there is no disruption which is an amino- termina truncatil ofon sequence thats overlap with part or all of at leas onet disrupted epitope region; (ii) 94-115 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 141- 153 of SEQ ID NO:1 or SEQ ID N():2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:I or SEQ ID NO :2; 179-191 of SEQ ID NO: 3; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2; and 210-218 of SEQ ID NO:3; and (iii) 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO: I or SEQ ID NO:2; 254-268 of SEQ ID NO: 1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO: 1 or SEQ ID NO:2; or the equivale regnt ion in a Shiga toxin A Subunit or derivative thereof. [ 19] In certain embodiments of Embodiment Set #1, the Shiga toxin effector polypeptide comprises a mutation, relat iveto a wild-type Shiga toxin A Subunit, in the B-cel and/orl T-cell epitope regio seln ect fromed the group of natively positione Shigad toxin A Subunit regions consisting of: (i) 1-15 of SEQ ID NO: 1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; and 53-66 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; or the equivale regint on in a Shiga toxin A Subunit or derivative thereof, wherein there is no disruption which is an ammo-termin truncaal tion of sequences that overlap with part or all of at leas onet disrupt edepitope region; (ii) 94-115 of SEQ ID NO: 1, SEQ IDNO:2, or SEQ IDNO:3; 141-153 ofSEQ IDNO:1 or SEQ IDNO:2; 140- 156 of SEQ IDNO:3; 179-190 ofSEQ IDNO:1 or SEQ IDNO:2; 179-191 ofSEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO: I or SEQ ID NO:2; and 210- 218 of SEQ ID NO:3; and (iii) 240-260 ofSEQ ID NO:3; 243-257 ofSEQ ID NO:1 or SEQ ID NO:2; 254-268 of SEQ ID NO: 1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 ofSEQ ID NO:3; and 285-293 ofSEQ ID NO:1 or SEQ ID NO:2; or the equivalent regio inn a Shiga toxin A Subunit or derivati thereve of,wherein there is no disruption which is an amino-termin truncaal tion of sequences that overlap with part or all of at least one disrupt edepitope region. . . . id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20"
[20] In certain embodiments of Embodiment Set «1, tire Shiga toxin effector polypeptide comprises a disruption of at leas onet endogenous epitope regio n selected from the group of natively positioned Shiga toxin A Subunits consist ingof: 94-115 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 ofSEQ IDWO 2016/196344 PCT/US2016/034778 9 NO:1 or SEQ IDNO:2; 140-156 ofSEQ IDNO:3; 179-190 ofSEQ IDNO:1 or SEQ ID NO:2: 179-191 ofSEQ ID NO:3; 204 ofSEQ ID NO:3: 205 ofSEQ ID NO: 1 or SEQ ID NO :2; or 210-218 of SEQ ID NO:3. [21 ] In certa embodimein nts of Embodiment Set # 1, the Shiga toxin effector polypeptide does not comprise a heterologous, MHC class !-restricte T-celd, l epitope. MHC class !-restricted, T-cell epitopes are known in the art or can be predicted by the skill edworke r.The term heterologous refers to MHC cla ssI- restricte T-celld, epitopes which are not natively prese ntin wild-type Shiga toxin A Subunits such, as, e.g., the wild-type Shiga toxin A Subunit which is most closely related to the Shiga toxin effector polypeptide of interest. 22. 22. 22. id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22"
[22] In certa embodimein nts of Embodiment Set #1, the Shiga toxin effector polypeptide comprises disruptions of at leas two,t thre four,e, five, six, seven, eight, or more endogenous, B-cell and/o T-cer ll epitope regions. 23. 23. 23. id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23"
[23] In certain embodimen ofts Embodiment Set # 1, one or more disruptions compris anes amino acid residue substitut ionrelat iveto a wild-type Shiga toxin A Subunit. 24. 24. 24. id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24"
[24] In certain embodiments of Embodiment Set #1, one or more endogenous, B- cell and/or T-cell epitope regions compris aes plurality of amino aci dresidue substitutions relative to a wild-type Shiga toxin A Subunit. . . . id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25"
[25] In certain embodimen ofts Embodiment Set #1, at leas one,t two, thre ore, four disruptions comprise a plurality of amino acid residue substitutions in the endogenous, B-cell and/o T-cer ll epitope regio relatn iveto a wild-type Shiga toxin A Subunit. 26. 26. 26. id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26"
[26] In certain embodimen ofts Embodiment Set«1, at lea stone disruptio n compris ates leas one,t two, thre four,e, five, six, seven, eight or, more amino acid residue substituti onsrelati tove a wild-type Shiga toxin A Subunit, and optiona lly wherein at leas onet substitution occurs at the native positly ioned Shiga toxin A Subunit amino aci dresidue selected form the group consisting of: 1 of SEQ ID NO: 1 or SEQ ID NO:2; 4 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 6 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID N():3; 8 of SEQ ID NO:I, SEQ ID NO:2, or SEQ ID NO:3; 9 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3: 11 ofSEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 12 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 33 of SEQ ID NO: 1 or SEQ ID NO:2; 43 of SEQ ID NO: 1 or SEQ ID NO:2; 44 of SEQ ID NO: 1 or SEQ ID NO:2; 45 of SEQ ID NO: 1 or SEQ IDWO 2016/196344 PCT/US2016/034778 NO:2; 46 of SEQ ID NO; 1, SEQ ID NO:2, or SEQ ID NO:3; 47 of SEQ ID NO: 1 or SEQ ID NO:2; 48 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3: 50 of SEQ ID NO: 1 or SEQ ID NO:2: 51 of SEQ ID NO: 1 or SEQ ID NO:2; 53 of SEQ ID NO: 1 or SEQ ID NO:2; 54 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 55 of SEQ ID NO: 1 or SEQ ID N():2: 56 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 57 of SEQ ID NO:1, SEQ ID XO:2 or SEQ ID NO:3; 58 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 59 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3: 60 of SEQ ID NO: 1 or SEQ ID NO:2; 61 of SEQ ID NO: I or SEQ ID NO:2; 62 of SEQ ID NO:1 or SEQ ID NO:2: 84 of SEQ ID NO: 1 or SEQ ID NO:2; 88 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3: 94 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 96 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 104 of SEQ ID NO: 1 or SEQ ID NO:2; 105 of SEQ ID NO: 1 or SEQ ID NO:2: 107 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3: 108 of SEQ ID NO:1 or SEQ ID NO:2; 109 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3: 110 of SEQ ID NO: 1 or SEQ ID NO:2; 111 of SEQ ID NO: i, SEQ ID NO:2, or SEQ ID N():3; 112 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141 of SEQ ID NO:L SEQ ID NO:2, or SEQ ID NO:3; 147 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 154 of SEQ ID NO: 1 or SEQ ID NO:2; 179 of SEQ ID NO: I, SEQ ID NO:2, or SEQ ID NO:3; 180 of SEQ ID NO: 1 or SEQ ID NO:2: 181 of SEQ ID NO: I or SEQ ID NO:2; 183 of SEQ ID NO: I, SEQ ID NO:2, or SEQ ID NO:3: 184 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3: 185 of SEQ ID NO: 1 or SEQ ID NO:2; 186 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 187 of SEQ IDNO:1 or SEQ IDNO;2; 188 of SEQ ID NO: I or SEQ ID NO:2; 189 of SEQ ID NO: I or SEQ ID NO:2: 197 of SEQ ID NO:3; 198 of SEQ ID NO: 1 or SEQ ID NO:2; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2; 247 of SEQ ID NO: I or SEQ ID NO:2; 247 of SEQ ID NO:3; 248 of SEQ ID NO: 1 or SEQ ID NO:2: 250 of SEQ ID NO:3; 251 of SEQ ID NO: I or SEQ ID NO:2: 264 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 265 of SEQ ID NO: 1 or SEQ ID NO:2; and 286 of SEQ ID NO: 1 or SEQ ID NO:2; or the equivalent amino acid residue in a Shiga toxin A Subunit or derivati therve eof In certain furthe embodr iment ats, leas twot disruptions each comprise at leas onet amino aci d residue substituti onsrelat iveto a. wild-type Shiga, toxin A Subunit selected form the group consisting of: 1 of SEQ ID NO: 1 or SEQ ID NO:2; 4 of SEQ ID NO: I, SEQ ID NO:2, or SEQ ID NO:3; 8 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 9WO 2016/196344 PCT/US2016/034778 11 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 11 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 33 of SEQ ID NO: 1 or SEQ ID NO:2; 43 of SEQ ID NO: 1 or SEQ ID NO:2; 45 of SEQ ID NO: 1 or SEQ ID NO: 2; 47 of SEQ ID NO: 1 or SEQ ID NO:2; 48 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQ ID NO: 1 or SEQ ID NO:2; 53 of SEQ ID NO: I or SEQ ID NO:2; 55 of SEQ ID NO: 1 or SEQ ID NO:2: 58 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 59 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 60 of SEQ ID NO: I or SEQ ID NO:2: 61 of SEQ ID NO: 1 or SEQ ID NO:2; 62 of SEQ ID NO: 1 or SEQ ID NO:2; 94 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 96 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3: 109 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; 110 of SEQ ID NO: 1 or SEQ ID NO:2; 112 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 147 of SEQ ID NO: L SEQ ID NO:2, or SEQ ID NO:3; 179 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 180 of SEQ ID NO:I or SEQ ID NO:2; 181 of SEQ ID NO: I or SEQ ID NO:2; 183 of SEQ ID NO I. SEQ ID SEQ ID NO 2. or SEQ ID NO:3; 184 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 185 of SEQ ID NO:1 or SEQ ID NO:2; 186 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 187 ofSEQ IDNO:1 or SEQ IDNO2; 188 of SEQ IDNOJ or SEQ IDNO:2: 189 of SEQ ID NO: 1 or SEQ ID NO:2; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2; 247 ofSEQ ID NO:1 or SEQ ID NO:2; 247 ofSEQ ID NO:3; 250 of SEQ ID NO:3; 264 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 265 ofSEQ ID NO: 1 or SEQ ID NO:2; and 286 of SEQ ID NO: 1 or SEQ ID NO:2; or the equivalent amino acid residue in a Shiga toxin A Subunit or derivati therve eof. 27. 27. 27. id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27"
[27] In certain embodiments of Embodiment Set #1, the Shiga toxin effector polypeptide comprises disruption of at leas thret endogene, ous, B-cell and/o T-celr l epitope regions selected from the group of consist ingof: (i) 1—15 of SEQ ID NO: 1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26 37 of SEQ ID N():3; 27 37 of SEQ ID NO: 1 or SEQ ID NO:2: 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; and 53-66 ofSEQ ID NO: 1, SEQ ID NO 2. or SEQ ID NO:3, or the equivalent region in a. Shiga toxin A Subunit or derivati thereve of,wherein there is no disrupti onwhich is an amino-terminal truncation of amino acid residues that overlap with part or all of at least one disrupted, endogenous, B-cell and/o T-cer ll epitope region; (ii) 94-115 of SEQ ID NO:I, SEQ ID NO:2, or SEQ ID NO:3; 141- 153 ofSEQ ID NO:1 or SEQ ID N():2; 140-156 ofSEQ ID NO: 3. 179-190 ofSEQ ID NO:1 or SEQ ID NO :2; 179-191 ofSEQ ID NO: 3; 204 ofSEQ ID NO: 3: 205 of WO 2016/196344 PCT/US2016/034778 12 SEQ ID NO; 1 or SEQ ID NO:2; and 210-218 of SEQ ID NO:3: and (iii) 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO; 1 or SEQ ID NO;2; 254-268 of SEQ ID NO: 1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO; 1 or SEQ ID NO;2; or the equivale regiont inn a Shiga toxin A Subuni tor derivati thereof,ve wherein there is no disrupti onwhich is a carboxy "terminal truncati ofon amino acid residues that overl withap part or all of at lea stone disrupted, endogenou B-cels, and/orl T-cell epitope and/or epitope region . 28. 28. 28. id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28"
[28] In certain embodiments of Embodiment Set #1, the Shiga toxin effector polypeptide comprises disruptions of at least two, endogenous, B-cell and/o T-cer ll epitope regions wherei, eachn disrupti oncomprises one or more amino aci dresid ue substitutions, and wherein the endogenous B-cell, and/or T-cell epitope regions are selected from the group of natively positioned Shiga toxin A Subunit regio ns consisting of; 3-14 of SEQ ID NO:3; 26 37 of SEQ ID NO:3: 27-37 of SEQ ID NO: I or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; or the equivalent regio inn a Shiga toxin A Subunit or derivative thereof. 29. 29. 29. id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29"
[29] In certain embodimen ofts Embodiment Set #1, the embedde ord inserted, heterologous T-ce,ll epitope does not disrupt any endogenous B-cell, and/or CD4+ T-cell epitope region described herein. . . . id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30"
[30] In certain embodimen ofts Embodiment Set #1, at leas onet disruptio n compris onees or more amino acid residue substitutions relat iveto a wild-type Shiga toxin A Subunit is selecte fromd the group consist ingof: D to A, D to G, D to V, D to L, D to I, D to F, D to S, D to Q, D to M, D to R, E to A, E to G, E to V, E to L, E to 1, E to F, E to S, E to Q, E to N, E to D, E to M, E to R, F to A, F to G, F to V, F to L, F to I, G to A, G to P, H to A, H to G, H to V, H to L, H to I, H to F, H to M, I to A, I to V, I to G, I to C, K to A, K to G, K to V, K to L, K to I, K to M, K to H, L to A, L to V, L to G, L to C, N to A, N to G, N to V, N to L, N to 1, N to F, P to A, P to G, P to F, R to A, R to G, R to V, R to L, R to I, R to F, R to M, R to Q, R to S, R to K, R to H, S to A, S to G, S to V, S to L, S to I, S to F, S to M, T to A, T to G, T to V, T to L, T to I, T to F, T to M, T to S, V to A, V to G, ¥ to A, Y to G, Y to V, Y to L, Y to 1, Y to F, Y to M, and Y to T. In certai furthern embodiments, the one or more amino acid residue substituti onsrelat iveto a wild-type Shiga toxin A Subuni t is selecte frdom the group consisting of: D to A, D to G, D to V, D to L, D to I, D to F, D to S, D to Q, E to A, E to G, E to V, E to L, E to 1, E to F, E to S, E to Q, E toWO 2016/196344 PCT/US2016/034778 13 N, E to D. E to M, E to R, G to A, H to A, H to G, H to V, H to L, H to I, H to F, H to M, K to A, K to G, K to V, K to L, K to I, K to M, K to H, L to A, L to G, N to A, N to G, N to V, N to L, N to I, N to F, P to A, P to G, P to F, R to A, R to G, R to V, R to L, R to I, R to F, R to M, R to Q, R to S, R to K, R to H, S to A, S to G, S to V, S to L, S to I, S to F, S to M, T to A, T to G, T to V, T to L, T to I, T to F, T to M, T to S, ¥ to A, Y to G, Y to V, Y to L, Y to 1, Y to F, and Y to M. 31. 31. 31. id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31"
[31] In certain embodimen ofts Embodiment Set # 1, at leas onet of the disraption(s compris) onees or more amino acid residue substitutions relati tove a wild-type Shiga toxin A Subunit selected from the group consist ingof: KI to A, G, V, L, I, F, M and H; T4 to A, G, V, L, I, F, M, and S; D6 to A, G, V, L, I, F, S, Q and R; S8 to A, G, V, I, L, F, and M; T9 to A, G, V, I, L, F, M, and S; S9 to A, G, V, L, I, F, and M; KI 1 to A, G, V, L, I, F, M and H; T12 to A, G, V, I, L, F, M, S, and K; S12 to A, G, V, I, L, F, and M; S33 to A, G, V, L, I, F, M, and C; S43 to A, G, V, L, I, F, and M; G44 to A or L; S45 to A, G, V, L, I, F, and M; T45 to A, G, V, L, I, F, and M; G46 to A and P; D47 to A. G. V. L. I. F, S, M. and Q; N48 to A. G. V. L, M and F; L49 to A, V, C, and G; Y49 to A, G, V, L, I, F, M, and T; F50 to A, G, V, L, I, and T; A51 ; D53 to A, G, V, L, I, F, S, and Q; V54 to A, G, I, and L; R55 to A, G, V, L, I, F, M, Q, S, K, and H; G56 to A and P; 157 to A, G, V, and M; L57 to A, V, C, G, M, and F; D58 to A, G, V, L, I, F, S, and Q; P59 to A, G, and F: E60 to A, G, V, L, I, F, S, Q, bi, D, M, T, and R: E61 to A, G, V, L, L F, S, Q, N, D, M, and R; G62 to A; R84 to A, G, V, L, I, F, M, Q, S, K, and H; V88 to A and G; 188 to A, V, C, and G; D94 to A, G, V, L, I, F, S, and Q; S96 to A, G, V, I, L, F, and M; T104 to A, G, V, L, I, F, M: and N; A105 to L; T107 to A, G, V, L, I, F, M, and P; S107 to A, G, V, L, I, F, M, and P; L108 to A, V, C, and G; S109 to A, G, V, I, L, F, and M; T109 to A, G, V, I, L, F, M, and S; GI10 to A; S I 12 to A, G, V, L, I, F, and M; DI 11 to A, G, V, R I, F, S, Q, and T; SI 12 to A, G, V, L, I, F, and M; D141 to A, G, V, L, I, F, S, and Q; G147 to A; V154 to A and G. R179 to A, G, V, L, I, F, M, Q, S, K, and H: T180 to A, G, V, L, I, F, M, and S; T181 to A, G, V, L, I, F, M, and S; DI 83 to A, G, V, L, I, F, S, and Q; DI 84 to A, G, V, L, I, F, S, and Q; LI85 to A, G, V and C; SI86 to A, G, V, I, L, F, and M; G187 to A: RI88 to A, G, V, L, I, F, M, Q, S, K, and H; S189 to A, G, V, I, L, F, and M; DI97 to A, G, V, L, I, F, S, and Q; D198 to A, G, V, L, I, F, S, and Q; R204 to A, G, V, L, I, F, M, Q, S, K, and H; R205 to A, G, V, L, I, F, M, Q, S, K and H; S247 to A, G, V, I, D F, and M; Y247 to A, G, V, L, I, F, and M: R248 to A, G, V, L, L F, M, Q, S, K, and H; R250 to A,G, V, L, I, F, M, Q, S, K, and H; R251 to A, G, V, L, I, F, M, Q, S, K, and H; D264 to A, G, V, L, I, F, S, and Q; G264 to A; and T286 to A, G, V, L, I, F, M, and S. 32. 32. 32. id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32"
[32] In certa embodiin ment of sEmbodiment Set #1, the Shiga toxi effectn or polypept ideconsis essts entia oflly the polypepti shownde in any one of SEQ ID NOs: 355-369 which further compris aes disruption of at leas one,t endogenous, B- cell and/or T-cell epitope region which does not overl withap an embedded or insert ed,heterologous CD8+, T-cel epitl ope. 33. 33. 33. id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33"
[33] In certa embodiin ment of sEmbodiment Set #1, the Shiga toxi effen ctor polypept idecompris ores consist esses ntia oflly the polypept ideshown in any one of SEQ ID NOs: 6-32, 340-354, and 370^138. 34. 34. 34. id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34"
[34] For certai embodimentn ofs Embodiment Set #1, the Shiga toxi effectn or polypepti isde capable of exhibiti ng(i) a catalytic activi levety comparal toble a wild-type Shiga toxi Aln fragme ornt wild-type Shiga toxin effector polypeptid e, (ii) a ribosome inhibition activ itywit ha half-maximal inhibitory concentration (IC50) value of 10,000 picomol orar less, and/or (iii) a significant level of Shiga toxi catn alyt acticivity. . . . id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35"
[35] For certai embodin ment of sEmbodiment Set #1, the Shiga toxi effen ctor polypept ideis capable of exhibiti ngsubcellular routing efficiency comparabl to ae wild-type Shiga toxi effen ctor polypept ideand/or capable of exhibiting a significant level of intracell routingular activity to the endoplasm retiic culum and/or cytosol from an endosomal start inglocat ionof a cell. 36. 36. 36. id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36"
[36] For certai embodin ment of sEmbodiment Set #1, the Shiga toxin effector polypepti isde capable of intracel luladeliveryr of the embedde ord insert ed, heterologous, T-cel epitopel from an early endosom compaal rtmen to a tMHC clas I s molecule of a cell in which the Shiga toxi effectn polypeptor ideis present For. certa furtherin embodiments the ,Shiga toxi effen ctor polypept ideis capable of exhibiting one or more Shiga toxi effen ctor functions in addition to the intracellul ar delivery, such as, e.g., the Shiga toxi effen ctor functions of: promot ingcellu lar internaliza directtion, ingsub-cellular routing to the cytos ol,ribosom inace tivati on, inducing caspase activity, causing cytostasi and/ors, causing cell death. In certain further embodiments, the heterologous, T-cel epitl ope is a CD8+ T-cel epitl ope, such as, e.g., with regar tod a human immune system. 37. 37. 37. id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37"
[37] In certai embodin ment ofs Embodiment Set #1, the Shiga toxin effector polypept idecomprises a disruption of at leas two,t endogenous epitope, regions -14-WO 2016/196344 PCT/US2016/034778 selected the group of natively positioned Shiga toxin A Subunit regions consist ing of: 94-115 ofSEQ ID NO:I, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190 ofSEQID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ IDNO:2; or210-218 ofSEQ ID NO:3; wherei then disrupti ondoes not consist sole lyof the amino acid residue substituti selecon ted from the group consisting of: S96Y of SEQ ID NO: 1 or SEQ ID NO:2; Y114S of SEQ ID NO: 1 or SEQ ID NO:2; R179A ofSEQ ID NO: 1 or SEQ ID NO:2; R179H ofSEQ ID NO: 1 or SEQ ID NO:2; L185A ofSEQ ID NO: 1 or SEQ ID NO:2; RI 88/1 ofSEQ ID NO: 1 or SEQ ID NO:2; R205A of SEQ ID NO: 1 or SEQ ID NO:2; R179A/R188A ofSEQ ID NO k or SEQ ID NO:2; or A188V ofSEQ ID NO:3. 38. 38. 38. id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38"
[38] In certain embodiments of Embodiment Set #1, the Shiga toxin effector polypeptide comprises a disruption comprisin a gmutati onof at leas onet amino acid residue in at lea stone, endogenous epitope regio selen cted from the group of natively positioned Shiga toxin A Subunit regions consist ingof: 1-15 of SEQ ID NO: I or SEQ ID NO:2; 3-14 ofSEQ ID NO:3: 26-37 ofSEQ ID NO:3; 27-37 of SEQ ID NO: 1 or SEQ ID NO 2: 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; wherein the Shiga toxin effecto polypeptider comprises no amino-terminus truncation overlapping wit hthe aforementioned disrupted epitope region. 39. 39. 39. id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39"
[39] In certain embodiments of Embodiment Set #1, the Shiga toxin effector polypeptide comprises a disruption comprisin a gmutati onof at leas onet amino acid residue in at lea sttwo, endogenous epitope regions selected from the group of natively positioned Shiga toxin A Subunit regions consist ingof: 1-15 of SEQ ID NO: 1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 ofSEQ ID NO: L SEQ ID NO:2, or SEQ ID NO:3; wherein the disruption does not consi stsole lyof the amino acid residue substitution R63W of SEQ ID NO: 1 or SEQ ID NO:2; and wherein the Shiga, toxin effector regio n compris noes amino-term inustruncation overlappi withng the aforementione two, d, disrupt edepitope regions. 40. 40. 40. id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40"
[40] In certa embodimein nts of Embodiment Set #1, the Shiga toxin effector polypeptide comprises a disruption comprisin a gmutation of at leas onet amino acid residue in at lea stone, endogenous epitope regio selecn ted from tire group of WO 2016/196344 PCT/US2016/034778 16 natively positioned Shiga toxin A Subunit regions consist ingof: 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO; 1 or SEQ ID NO;2: 254-268 of SEQ ID NO; I or SEQ ID NO:2: 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO: 1 or SEQ ID NO:2; wherein the Shiga toxin effecto polypeptr ide compris noes carboxy-termi truncationnus overlapping wit hthe aforementi oned disrupted epitope region. 41. 41. 41. id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41"
[41] In certain embodiments of Embodiment Set #1, the Shiga toxin effector polypeptide comprises a disruption comprisin a gmutati onof at lea stone amino acid residue in at lea sttwo, endogenous epitope regions selected from the group of natively positioned Shiga toxin A Subunit region conss ist ingof: 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO: I or SEQ ID NO:2; 254-268 of SEQ ID NO: 1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO: 1 or SEQ ID NO:2: wherei then Shiga toxin effector polypept ide does not comprise the mutati onselect fromed the group consist ingof: R248H of SEQ ID NO: 1 or SEQ ID NO;2; A250V of SEQ ID NO: 1 or SEQ ID NO:2; R251H of SEQ ID NO: 1 or SEQ ID NO:2; A253G of SEQ ID NO; 1 or SEQ ID NO:2; S254T of SEQ ID NO: 1 or SEQ ID NO:2: C261A of SEQ ID NO: 1 or SEQ ID NO:2; R289K of SEQ ID NO: I or SEQ ID NO:2; R248H and R251H of SEQ ID NO; 1 or SEQ ID NO;2: A253G and S254T of SEQ ID NO; 1 or SEQ ID NO;2; the deletion of S247-M252 of SEQ ID NO: 1; S246F of SEQ ID NO:3; A282V of SEQ ID NO:3; 1291V of SEQ ID NO:3; S246F of SEQ ID NO:3; and where thein Shiga toxin effector polypeptide compris noes carboxy-termi truncationnus overlapping with the aforementione two,d, disrupted epitope regions. 42. 42. 42. id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42"
[42] In certain embodiments of Embodiment Set «1, the de-immunized, Shiga toxin effector polypeptide compris ores consis essts entiall of they polypept ide shown in any one of SEQ ID NOs: 6 27. 29 32. 340-354, and 370-438. 43. 43. 43. id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43"
[43] In certain embodiments of Embodiment Set #1, the Shiga toxin effector polypeptide of the present invent oni compris onees or more mutations relat iveto a naturally occurri Ang Subunit of a member of the Shiga, toxin family which changes an enzymat activityic of the Shiga toxin effector polypeptide the ,mutation select ed from at leas onet ammo acid residue deletion, insertion, or substituti on.In certai n furthe embodimer nts, the mutation relat iveto the natura occurrilly Ang Subunit reduc esof eliminat aes cytotoxic activity of the Shiga toxin effector polypeptide but the Shiga toxin effecto polypeptr ideretains at leas onet othe Shigar toxin effectorfunction, such as, e.g., promot ingcellular internaliza and/ortion direct ing intracellul routaring to a certa subcellin ular compartment(s In certai). furthen r embodiments the ,mutati onrelative to the natural occurrly ingA Subunit is select ed from at leas onet amino aci dresidue substitution, such as, e.g., A23 IE, R75 A, Y77S, Y114S, E167D, R170A, R176K, and/or W203A in SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3. 44. 44. 44. id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44"
[44] In certai embodimn ents of Embodiment Set #1, the Shiga toxin effector polypept idecomprises (i) a Shig atoxin Al fragment derived region having a carboxy terminus and (ii) a disrupt edfurin-cleava motige atf the carboxy-term inus of the Al fragme regint on. In certa furthein embodimr ents, the disrupt edfurin­ cleavage mot ifcomprises one or more mutations, relat iveto a wild-type Shiga toxin A Subunit, the mutati onalteri atng leas onet amino acid residue in a region natively positioned at 248-251 of the A Subunit of Shiga-like toxi 1n (SEQ ID NO:1) or Shiga toxin (SEQ ID NO:2), or at 247-250 of the A Subunit of Shiga-like toxin (SEQ ID NO:3); or the equivale regionnt in a Shiga toxi An Subuni tor derivative thereof. In certai furthern embodiment thes, disrupt edfurin-cleav motiage f comprises one or more mutations, relati tove a wild-type Shiga toxi An Subunit, in a minimal furin cleavage sit eof the furin-cleava motgeif. In certai furthen r embodiments, the minimal furin cleavag sitee is represented by the consensu amis no acid sequence R/Y-x-x-R and/or R-x-x-R. 45. 45. 45. id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45"
[45] In certai embodin ment ofs Embodiment Set #1, the Shig atoxin effector polypeptide compris (i)es a Shiga toxi Aln fragment derived region having a carboxy terminus and (ii) a disrupted furin-cleava motge ifat the carboxy-termi nus of the Al fragme regint on. In certa furtherin embodiments the ,disrupt edfurin­ cleavage moti comprisf anes amino acid residue substitution in the furin-cleavage mot ifrelati tove a wild-type Shiga toxin A Subunit. In certa furtherin embodiments , the substitution of the amino acid residue in the furin-cleavage mot ifis of an arginine residue wit ha non-positivel charged,y amm oaci dresidue selected from the group consist ingof: alanine glycin, proline,e, serin e,threon ine,aspartat e, asparagine, glutamat glutame, ine, cysteine, isoleucine leuc, ine, methioni ne,valine, phenylalani tryptophan,ne, and tyrosi ne.In certai embodimn ents, the substitution of the amino aci dresidue in the furin-cleavage mot ifis of an arginine residue wit ha histidine. -17-WO 2016/196344 PCT/US2016/034778 18 46. 46. 46. id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46"
[46] In certain embodiments of Embodiment Set #1, die Shiga toxin effector polypepti eithede, aloner or as a component of a first cell-targeti moleculng ise, capable when introduced to cells of exhibiting cytotox kirt comparable to the cytotoxic ofity a wild-type Shiga, toxin A1 polypeptide and/or second cell-targetin g molecule consist ingof the firs cell-t target molecing ule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin Al fragment. 47. 47. 47. id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47"
[47] For certain embodimen ofts the Shiga toxin effector polypeptide of Embodiment Set #1, a cell-targetin molecg ule of the prese ntinvention comprisi ng the Shiga toxin effecto polypeptider is capable when introduc toed a chordate of exhibiting improved, in vivo tolerabili comparedty to a. second cell-targetin g molecule consist ingof the firs cell-t target moleculing excepte for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin Al fragment and/or wild-type Shiga, toxin form-cleava sitege at the carboxy terminus of its Ai fragment region. 48. 48. 48. id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48"
[48] In certain embodiments of Embodiment Set #1, tire Shiga toxin effector polypeptide of the present invention compris aes Shiga toxin effector regio derivedn from ammo acids 75 to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. 49. 49. 49. id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49"
[49] In certain embodiments of Embodiment Set #1, the Shiga toxin effector polypeptide of the present invention compris aes Shiga toxin effector region derived from amino acids 1 to 241 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certa furtherin embodiment thes, Shiga toxin effector region is derived from amino acid s1 to 251 of SEQ ID NO: I, SEQ ID NO:2, or SEQ ID NO:3. In certa furtherin embodiments, the Shiga toxin effector region is derived from amino acids 1 to 261 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.
Embodiment Set #2 - Cell-Target Moleculeing Comprising a De-immunized Shiga Toxin Effect Polypeptideor Comprising an Embedded or Inserted. Heterologous, T- Cell Epitope and A Non-overlapping De-immunized Sub-Region 50. 50. 50. id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50"
[50] The present invention provides cell-targeti molengcule eacs, hcomprising (i) a bindin gregion capable of specificall bindingy an extracel targelular biomolt ecul e and (ii) a de-immunize Shigad, toxin effecto polypeptider of Embodiment Set #1 (see e.g. Figure 1, depicting illustrati examve ples of four exemplary, embodiments of the cell-targeting molecule ofs this embodime setnt #2). For example, certa inWO 2016/196344 PCT/US2016/034778 19 embodiments of set #2 is the cell-targeti moleculeng comprisin (i)g a bindin gregion capable of specifica bindinglly an extracel targlular biomoleculet ande (ii) a de- immunized, Shiga toxin effector polypeptide comprisin atg lea stone insert ored embedded, heterologous epitope (a) and at leas onet disrupted, endogenous, B-cell and/o CD4+r T-cel epitopel region (b), wherein the heterologous epitope does not overlap with the embedde ord inserte heted, rologous T-ce,ll epitope. For certain further embodiments, the Shiga toxin effecto polypeptider is capabl ofe exhibiting at leas onet Shiga toxin effector function, such as, e.g., directing intracellul routingar to tire endoplasmic reticulum and/or cytosol of a cell in which tire polypeptide is present, inhibiting a ribosome function, enzymatically inactiva tinga ribosome , causing cytostasi and/ors, causing cytotoxicity. In certain further embodiments, the heterologous T-ce,ll epitope is a CD8+ T-cell epitope, such as, e.g., wit hregard to a human immune system. For certai furthern embodiments, the heterologous T-ce,ll epitope is capable of being presented by a MHC class I molecule of a cell In. certain further embodiments, the cell-targeting molecule of the prese ntinvention is capable of one or more the following; entering a cel l,inhibiting a ribosome function, causing cytostasi causings, cel death,l and/or deli vering the embedded or inserted , heterologous T-ce,ll epitope to a. MHC class I molecule for presentat onion a cellular surface. For certa furthein embodimer nts, the cell-targeting molecule is capable when introduced to cel lsof exhibiting a cytotoxic compaity rable or better than a referenc molecule suche, as, e.g,, a second cell-targeting molecule consisting of the cell-targetin molegcule except for all of its Shiga toxin effector polypeptide component(s) eac hcomprise a wild-type Shiga toxin Al fragment. 51. 51. 51. id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51"
[51] For certain embodimen ofts Embodiment Set #2, the cell-target moleingcule compris aes molecular moiety located carboxy-termina to the carboxy-l terminus of the Shiga toxin Al fragment region. 52. 52. 52. id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52"
[52] For certain embodimen ofts Embodiment Set #2, the cell-target moleingcule of the prese ntinvention is capabl whene introduce to da chordate of exhibiting improved in vivo tolerabili and/oty star bilit compay red to a refere ncemolecule, such as, e.g., a second cell-target moleingcule consist ingof the ceil-target molecing ule except for all of its Shiga toxin effecto polypeptider component(s) eac hcomprise a wild-type Shiga toxin A l fragment and/or wild-type Shiga toxi furn in-cleavage site at the carboxy termin ofus its Al fragment region. In certa furthein embodiments,rthe Shiga toxin effector polypept ideis not cytotoxic and the molecula moietr isy cytotoxic. 53. 53. 53. id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53"
[53] In certain embodiment of sEmbodiment Set #2, the binding region and Shiga toxi effectorn polypeptide are linked together eit,her direct orly indirectly. 54. 54. 54. id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54"
[54] In certa embodimein nts of Embodiment Set #2, the binding region comprises a polypept idecomprising an immunoglobulin-t bindiypeng region. In certai furtn her embodiments the ,binding region comprises a polypept ideselect fromed the group consisti of:ng an autonomous Vh domai n,single-domain antibody fragme (sdAb),nt nanobod heavyy, chain-antibody domain derived from a cameli (VHHd or Vh domain fragment), heavy-chain antibody domain derived from a cartilagi nousfish (VHH or Vr domain fragment), immunoglobuli newn antigen recept (IgNAor R), Vnar fragment, single-chain variabl fragmee (scFv),nt antibody variabl fragme ent (Fv), complement determary ini regionng 3 fragme (CDR3)nt , constrai FR3-ned CDR3-FR4 polypeptide (FR3-CDR3-FR4), Fd fragment, small modular immunopharmaceuti (SMIP)cal domain, antigen-bindi fragmeng (Fab),nt Armadil lo repeat polypept ide(ArmRP), fibronectin-deri 10thved fibronecti typen III domai n (10Fn3), tenasci typen III domain (TNfn3), ankyrin repea motit domaif n,low- density-lipoprotein-receptor-de A-domairived (LDLn R-A), lipocal (antiin cal in), Kunit zdomai n,Protein-A-deri Zved domain, gamma -Bcrystalline-de domairivedn, ubiquitin-derived domain, Sac7d-derived polypept ide(affiti n),Fyn-derive SH2d domain, miniprotei C-tn,ype lectin-l domainike scaffold, engineered antibody mimic, and any genetically manipula tedcounterp artsof any of the foregoing whic h retai bindingn functionality. 55. 55. 55. id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55"
[55] For certai embodin ment ofs Embodiment Set #2, the cell-targeti molengcule of the prese ntinventio is ncapable of exhibiting (i) a catalyt activic itylevel comparabl to ae wild-type Shig atoxin Al fragme ornt wild-type Shiga toxin effector polypepti (ii)de, a ribosome inhibition activi witty ha half-maxim inhibial tory concentrati (IC50)on value of 10,000 picomola or rless, and/or (iii) a significant leve l of Shig atoxin catalyt actiic vity. 56. 56. 56. id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56"
[56] For certain embodiment ofs Embodiment Set #2, the cell-targeti molecng ule of the present invention and/or its Shiga toxin effector polypept ideis capable of exhibiting subcellula routir ng efficiency comparab tole a referenc cele l-target ing molecule comprising a wild-type Shiga toxin Al fragment or wild-type Shiga toxin effector polypept ideand/or capable of exhibiting a significant leve ofl intracellu lar -20-routing activ ityto the endoplasm retiic culum and/or cytosol from an endosoma l starti locating ofon a cell. 57. 57. 57. id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57"
[57] For certain embodiments of Embodiment Set #2, where byadministration of the cell-targeting molecule of the present invention to a cell physical coupledly wit h the extracell targetular biomolecule of the cell-targeti molecng ule’s binding region, the cell-target molingecule is capable of causing deat ofh the cel l.In certain furthe r embodiments admi, nistrati of theon cell-targeti molngecule of the invention to two differe populatnt ions of cell types which differ wit hrespect to the presence or level of the extracell־ targetular biomolecule, the cell-targeti molngecule is capable of causing cell deat toh the cell-types physical couplely witd han extracell targetular biomolecule of the cytotoxic cell-target moleingcule’s binding region at a CD50 at least three times or less than the CD5q to cell types which are not physicall coupledy with an extracell targetular biomolecule of the cell-targeti moleculng ’s bindinge region. For certa embin odiments whereby, administrati of theon cell-targeting molecul ofe the present inventi toon a first population of cell whoses members are physical coupledly to extracell targetular biomolecules of the cell-target ing molecule’s binding region, and a second population of cell whoses members are not physicall coupledy to any extracellu targetlar biomolec uleof the binding region, the cytotoxi effectc of the cell-targeti molngecule to members of said first populat ionof cell rels ati tove members of sai dsecond population of cell iss at leas 3-foldt greater.
For certa embodin iments where, byadministrati of theon cell-targeti molngecule of the present inventio to na first populations of cell whoses members are physical ly coupled to a significant amount of the extracel targetlular biomolec uleof the cell­ target moleing cule’s binding region, and a second populat ionof cel lswhose members are not physical coupledly to a significa amountnt of any extracell ular target biomolecule of the binding region, the cytotoxic effect of the cell-target ing molecule to members of said first population of cell relats iveto member ofs said second populatio ofn cell iss at lea st3-fold great er.For certai embodimn ents, where byadministration of the cell-targeti molecng ule of the prese ntinvention to a firs populatt ion of target biomolecu posile tive cell ands, a second population of cell s whose members do not express a significant amount of a target biomolecule of the cell-targeti molngecule’s binding region at a cellular surfac thee, cytotoxic effect of the cell-targeti molngecule to members of the first populati ofon cell rels ati tove members of the second population of cel lsis at least 3-fold greater. -21-WO 2016/196344 PCT/US2016/034778 22 58. 58. 58. id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58"
[58] For certain embodimen ofts Embodiment Set #2, the cell-target moleingcule of the prese ntinvention is capabl whene introduce to dcells of exhibiting a cytotoxic witity ha half-maxima inhibl itor concentry ati (CD50)on value of 300 nM or less and/o capar ble of exhibiting a significa levent ofl Shiga toxin cytotoxicity. 59. 59. 59. id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59"
[59] For certa embodin imen ofts Embodiment Set #2, the cell-target moleingcule of the prese ntinvention is capabl ofe deliver ingan embedded or inserte d, heterologous CD8+, T-cell epitope to a MHC class I presentati pathwayon of a cell for cell-surfa presce entat ofion the epitope bound by a MHC clas I smolecule. 60. 60. 60. id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60"
[60] In certain embodimen ofts Embodiment Set #2, tire cell-target moleculeing compris aes molecular moiety associated with the carboxy-terminus of the Shiga toxin effector polypeptide. In certai embodimen nts, the molecul moiety'ar comprise s or consists of the bindin gregion In. certai embodimen nts, the molecul moiear ty compris ates lea stone amino acid and the Shiga toxin effecto polypeptider is linked to at leas onet amino acid residue of the molecular moiety In. certai furthen r embodiments, the molecular moiet andy the Shiga toxin effector polypeptide are fused forming a continuous polypeptide. 61. 61. 61. id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61"
[61] In certain embodimen ofts Embodiment Set #2, the cell-target moleculeing furthe comprisr aes cytotoxic molecul moietyar associate withd the carboxy- termin ofus the Shiga toxin effecto polyper ptide For. certain embodiments, the cytotoxic molecular moiety is a cytotoxic agent, such as, e.g., a smal moleculel chemotherapeutic agent, anti-neoplastic agent, cytotoxic antibiotic, alkyla tingagent, antimetabolite topoisom, eras inhibitore and/or, tubulin inhibitor known to the skill ed worker and/or describe hereid n.For certai furthen embodimer nts, the cytotoxic molecular moiety is cytotoxic at concentra oftions les thans 10,000, 5,000, 1,000, 500, or 200 pM. 62. 62. 62. id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62"
[62] In certain embodiments of Embodiment Set #2, the binding region is capable of binding to an extracellula targe rbiomolecult selece ted from the group consist ing of: CD20, CD22, CD40, CD74, CD79, CD25, CD30, HER2/neu/ErbB2, EGFR, EpCAM, EphB2, prostate-specific membrane antige Cripto,n, CDCP1, endoglin, fibroblas activatt protein,ed Lewis-Y, CD19, CD21, CS1/ SLAMF7, CD33, CD52, CD133, CEA, gpA33, mucin, TAG-72, tyrosine-pr oteinkinase transmembrane receptor (RORl or NTRKR1), carbonic anhydrase IX, folate bindin proteg in, gangliosi GD2,de gangliosi GD3,de ganglioside GM2, ganglioside Lewis-Y2, VEGFR, Alpha Vbeta3, AlphaSbetal, ErbBl/EGFR, Erb3, c-MET, IGF1R, EphA3, WO 2016/196344 PCT/US2016/034778 23 TRAIL-RI, TRAIL-R2. RANK, FAP, tenascin, CD64, mesothel BRCA1,in, MART-l/MelanA, gplOO, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, GAGE- 1/2, BAGE, RAGE, NY-ESO-1, CDK-4, beta-catenin, MUM-1, caspase -8, KIAA0205, HPVE6, SART-1, FRAME, carcinoembry antigen,onic prostat speecific antige prostan, stemte cell antigen, huma naspartyl (asparaginyl) beta-hydroxyla se, EphA2, HER3/ErbB-3, MUC1, MART-l/MelanA, gplOO, tyrosinase associa ted antigen, HPV-E7, Epstein-Bar virusr antigen, Bcr-Abl, alpha-fetoprote antigein n, 17-Al, bladder tumor antigen, CD38, CD15, CD23, CD45 (protein tyrosine phosphatas receeptor type C), CD53, CD88, CD129, CD183, CD191, CD193, CD244, CD294, CD305, C3AR, FceRIa, galectin-9, IL-1R (interleu1kin- receptor ), mrp-14, NKG2D ligand, programmed death-ligand 1 (PD-L1), Siglec-8, Siglec-10 , CD49d, CD13, CD44, CD54, CD63, CD69, CD123, TLR4, FceRIa, IgE, CD107a, CD203c, CD14, CD68, CD80, CD86, CD105, GDI 15, F4/80, ILT-3, galecti n-3, GDI la-c GITR, L, MHC class I molecule, MHC clas IIs molecule (optionally complexed with a peptide) CD284, (TLR4), CD107-Mac3, CD195 (CCR5), HLA- DR CD16/32, CD282 (TLR2), GDI 1c, and any immunogenic fragment of any of the foregoing. 63. 63. 63. id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63"
[63] In certa embodimein nts of Embodiment Set #2, the binding regio isn linked, either directly or indirectly, to the Shiga toxin effecto polypeptider by at leas onet coval entbond which is not a disulfide bond. In certai furthen embodimer nts, the bindin gregio isn fused, either direct orly indirectl to y,the carboxy -terminus of the Shiga toxin effector polypeptide to form a single, continuous polypepti de.In certain furthe embodr iment thes, bindin regiog isn an immunoglobulin-type binding region. 64. 64. 64. id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64"
[64] In certain embodiments of Embodiment Set «2, the disrupted furin-cleavage motif compris onees or more mutations in the minima l,furin-cleavage sit erelat iveto a wild-type Shiga toxin A Subunit. In certain embodiments, the disrupt edfurin- cleava motifge is not an ammo-terminal truncati ofon sequences that overlap with part or all of at leas onet amino acid residue of the minimal furin-cleava site.ge In certa embodimein nts, the mutati onin the minima l,furin-cleavage sit eis an amino acid deleti on,insertion, and/or substitution of at leas onet amino acid residue in the R/Y-x-x-R furi ncleavage motif. In certai furthen embodimer nts, the disrupted furin-cleavage motif compris ates leas onet mutati onrelat iveto a wild-type Shiga toxin A Subunit, the mutation alter ingat lea stone amino acid residue in the regio n natively positioned 1) at 248-251 of the A Subunit of Shiga-like toxin 1 (SEQ IDWO 2016/196344 PCT/US2016/034778 24 NO: 1) or Shiga toxin (SEQ ID NO: 2), or 2) at 247-250 of the A Subunit of Shiga - like toxin 2 (SEQ ID NO:3), or the equivalent amino acid sequence position in any Shiga toxin A Subunit. In certain further embodiments, the mutati onis an amino acid residue substitution of an arginine residue with a. non-positively charged, amino acid residue. 65. 65. 65. id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65"
[65] In certain embodimen ofts Embodiment Set #2, the cell-target moleingcule of the prese ntinvention is capable when introduc toed cells of exhibiting cytotoxic ity comparable to a cytotoxicity of a reference molecule, such as, e.g., a second cell- targeting molecule consisting of tire cell-targeti molecng ule except for all of its Shiga toxin effector polypeptide component(s each) compris ae wild-type Shiga toxin A1 fragment. 66. 66. 66. id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66"
[66] In certain embodiments of Embodiment Set #2, the bindin regg ion comprise s the peptide or polypept ideshown in any one of SEQ ID NOs: 83339־. In certain further embodiment thes, bindin regiog comprisn ores consists essentia oflly the polypeptide represented by any of the following: amino acid s1-245 of any one of SEQ ID NOs: 33, 64, and 65; 269-513 of SEQ ID NO:40 or SEQ ID NO:80; ammo acid 269-520s or 269-521 of any one of SEQ ID NOs: 36, 66, and 67; amino acids 1־232, 1־233, 1-234, 1-235, 1-236, 1-242, 1-243, 1-244, 1-245, 1-246, 1-252, 1- 253, 1-254, 1-255, or 256 ؛ of any one of SEQ ID NOs: 47-119 and 176-248; amino acids 269-498 or 269-499 of any one of SEQ ID NOs: 37-39, 68-79, and 81; amino acids 269-499, 269-512, 269-513, or 280-510 of any one of SEQ ID NOs: 34, 35, 41-56, and 82. 67. 67. 67. id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67"
[67] In certain embodimen ofts Embodiment Set #2, tire cell-target moleingcule of tire prese ntinvention compris ores consis essts entially of the polypeptide shown m any one of SEQ ID NOs: 43-62, 64-82, and 439-513. 68. 68. 68. id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68"
[68] In certain embodiments of Embodiment Set #2, the bindin regg ion sterically cove rsthe carboxy-terminus of the Al fragment region. 69. 69. 69. id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69"
[69] In certain embodimen ofts Embodiment Set #2, the molecular moiety sterically cover thes carboxy-terminus of the Al fragment region. In certa furthein r embodiments, the molecular moiety comprises the bindin regiong . 70. 70. 70. id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70"
[70] In certain embodimen ofts Embodiment Set #2, tire cell-target moleingcule of the prese ntinvention compris aes binding region and/o moleculr moiear ty' locat ed carboxy-termina to the lcarboxy-terminus of the Shiga toxin Al fragment region. In certain further embodiment tires, mass of the bindin regg ion and/o molecr ularmoiet isy at leas 4.5t kDa, 6, kDa ,9 kDa, 12 kDa ,15 kDa, 20 kDa ,25 kDa ,28 kDa, kDa, 41 kDa, 50 kDa, 100 kDa, or greater. 71. 71. 71. id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71"
[71] In certa embodimein nts of Embodiment Set #2, the cell-targeti molengcule compris aes binding region wit ha mass of at leas 4.5t kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeti molngecule retai thens appropriate leve ofl the Shiga toxin biological activ itynoted herei (e.g.,n cytotoxici and/orty intracel lularoutirng). 72. 72. 72. id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72"
[72] In certa embodiin ment of sEmbodiment Set #2, the binding region is comprised within a relativel large,y molecular moiet compy rising such as, e.g., a molecular moiety wit ha mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or great er,as long as the cell-targeti molngecule retains the appropri ateleve ofl the Shiga toxi biologin cal activity noted herein. 73. 73. 73. id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73"
[73] In certa embodiin ment of sEmbodiment Set #2, the amino-terminus of the Shiga toxin effector polypept ideis at and/or proxim toal an amino-terminus of a polypept idecomponent of the cell-targeti molengcule In. certain furthe r embodiments, the binding region is not located proxim toal the amino-termin of us the cell-target molingecule relat toive the Shiga toxi effectorn polypeptid Ine. certai furthern embodiments the, binding region and Shiga toxi effen ctor polypeptide are physical arrangely or doriented within the cell-target molingecule such that the binding region is not locat proximaed tol the amino-term inusof the Shiga toxin effector polypeptide In .certai furthen embr odiments the ,binding region is located within the cell-targeti molengcule more proxim toal the carboxy-term ofinus the Shiga toxi effectn polypeptior thande to the amino -terminus of the Shiga toxin effector polypeptide For. certai furthen embodimr ents, the cell-targeti molecng ule of the prese ntinvention is capable when introduced to cell ofs exhibiting cytotoxici thatty is greater than that of a third cell-target molingecule having an amino-term inusand comprising the binding region and the Shiga toxi effen ctor polypept idewhic his not positioned at or proxim toal the amino-terminus of the third cell-targeti molecng ule. For certain furthe embr odiments the ,cell-targeti ng molecule of the present inventio exhibin tscytotoxi wicitthy better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fol d,9-fol d,or greater cytotoxi city as compared to the cytotoxic ofity the third cell-targeti molecng ule. For certai n furthe embr odiments the ,cytotoxici of thety cell-targeti molecng ule of the present -25-invention to a populat ionof target positive cel lsis 3-fold, 4-fol d,5-fol d,6-fold, 7­ fold, 8-fol d,9-fol d,10-fold or greater than the cytotoxi ofcit they third cell-targeti ng molecule to a secon populatd ion of target positive cel lsas assayed by CD50 values .
In certa furtin her embodiments, the third cell-targeti molngecule does not compri se any carboxy-term endoplinal, asm retiic culum retention/retriev signal almoti off the KDEL family. ׳ 74. 74. 74. id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74"
[74] In certai embodin ment ofs Embodiment Set #2, the amino-termi ofnus the Shiga toxi effectorn polypept ideis at and/or proxim toal an amino-termin ofus a polypept idecomponent of the cell-targeti molengcule. In certai furthern embodiments the ,binding region is not located proxim toal the amino-termi ofnus the cell-target molingecule relat toive the Shiga toxin effector polypeptide In . certai furthern embodiments the ,binding region and Shiga toxi effen ctor polypept ide are physicall arrany gedor oriented within the cell-target molingecule such that the binding region is not locat proximed toal the amino-termi ofnus the Shiga toxin effector polypeptide In .certa furtherin embodiments, the binding region is located withi then cell-target molingecul moree proxim toal the carboxy-term ofinus the Shiga toxi effen ctor polypept idethan to the amino-termi ofnus the Shiga toxin effector polypeptide For. certa furtin her embodiments the ,cell-target molecing ule of the present invention is not cytotoxic and is capable when introduced to cell ofs exhibiting a great subceler lula routingr efficiency from an extracell spaceular to a subcellular compartme ofnt an endoplasm reticic ulum and/or cytos asol compared to the subcellular routing efficiency of a third cell-targeti molecng ule having an amino­ terminus and comprising the binding region and the Shig atoxin effector polypept ide which is not positioned at or proxim toal the amino-termi ofnus the third cell­ target moleing cule In. certa furthein embodimr ents, the third cell-target molecing ule does not comprise any carboxy-termi endoplanal, smic reticulum retention/ret rieval signa motl ifof the KDEL family. 75. 75. 75. id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75"
[75] In certa embodiin ment ofs Embodiment Set #2, the amino-terminus of the Shiga toxin effector polypeptide is at and/or proxim toal an amino-termi ofnus a polypepti componentde of the cell-targeti molengcule In. certai furthen r embodiments, the binding region is not located proxim toal the amino-termi ofnus the cell-target moleingcule relat toive the Shiga toxi effen ctor polypeptide. In certai furthern embodiments the ,binding region and Shiga toxin effect polypeptor ide are physical arrangedly or oriented within the cell-targeti molngecule such that the -26-binding region is not located proxima to lthe amino-term inusof the Shiga toxin effector polypeptide In .certa furtherin embodiments the ,binding region is located within the cell-targeti molecng ule more proxima to lthe carboxy-term ofinus the Shiga toxin effector polypept thanide to the amino-termi ofnus the Shig atoxin effector polypepti de.For certai furthern embodiments the ,cell-targeti molengcule of the present invention exhibit lows cytotoxic potency (i.e. is not capabl whene introduced to certai positin ve target cell types of exhibiting a cytotoxic greatity er than 1% cell death of a cell population at a cell-target molecing ule concentrat of ion 1000 nM, 500nM, 100 nM, 75 nM, or 50 nM) and is capabl whene introduce tod cell ofs exhibiting a greater subcellula routingr efficiency from an extracellular spac toe a subcellula compar rtmen of ant endoplasm reticic ulum and/or cytosol as compared to the subcellular routing efficiency of a third cell-targeti molengcule having an amino-term inusand comprising the binding region and the Shiga toxi n effect polypeptor idewhich is not positioned at or proxim toal the amino-termin ofus the third cell-target molecing ule. In certai furthern embodiments the ,thir celld ­ target molecing ule does not compri seany carboxy-termi endoplasmnal, retiic culum retention/retrie signalval mot ifof the KDEL family. 76. 76. 76. id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76"
[76] In certai embodin ment of sEmbodiment Set #2, the cell-targeti molngecule of the present invention or a, polypepti compde onent thereof, comprises a carboxy- termina endopll, asm retiic culum retention/retri signevalal moti off a member of the KDEL family. For certain further embodiments the ,carboxy-termina endoplasml ic reticulum retention/ret signrievalal mot ifis selected from the group consisti of:ng KDEL, HDEF, HDEL, RDEF, RDEL, WDEL, YDEL, HEEF, HEEL, KEEL, REEL, KAEL, KCEL, KFEL, KGEL, KHEL, KLEL, KNEL, KQEL, KREL, KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL, KDEF, KKEL, HADL, HAEL, HIEL, HNEL, HTEL, KTEL, HVEL, NDEL, QDEL, REDL, RNEL, RTDL, RTEL, SDEL, TDEL, SKEL, STEL, and EDEL. In certai furthern embodiments, the cell-targeting molecul ofe the present invention is capable when introduced to cell ofs exhibiting cytotoxic thatity is greater than that of a fourth cell-targeting molecule consisting of the cell-targeti molngecule excep fort it does not compri se any carboxy-terminal endoplasm, retiic culum retention/ret sigrienalval moti off the - KDEL family. In certai furthern embodiments the ,cell-target molecing ule of the present inventi ison capable of exhibiti nga cytotoxi witcity hbetter optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fol d,6-fol d,9-fold, or greater cytotoxicity -27-as compared to a reference molecule, such as, e.g., the fourth cell-targeti ng molecule In. certa furtherin embodiments, the cytotoxici of thety cell-target ing molecule of the prese ntinvention to a population of target positive cells is 3-fold, 4­ fold, 5-fold 6-fold,, 7-fold, 8-fol d,9-fol d,10-fol ord great thaner the cytotoxi ofcit y the fourt cellh -targeti molengcule to a second population of target positive cell ass assayed by CD50 values.
Embodiment Set #3 - Cell-Targeti Moleng cul Comprie sin ag Carboxy-Termi nal Endoplasm Retiic culum Retention/Retri Signevalal Moti andf a Shiga Toxin Effector Polypepti Compride sin ang Embedded or Inserted, Heterologous, T-Cel l Epitope 77. 77. 77. id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77"
[77] The present inventio provin des cell-targeti molengcules each, comprising (i) a binding region capable of specifical bindingly an extracell targetular biomolecule; (ii) a Shig atoxin effector polypept idecomprising an inserted or embedded, heterolog epitousope; and (iii) a carboxy-terminal endoplasm, retiic culum retention/retri signevalal moti f.In certa embodimin ents, the cell-target molingecule of the present invention comprises (a) a binding region capable of specificall y binding at leas onet extracell targetular biomolecule; (b) a Shiga toxi effecn tor polypeptide comprising an embedded or insert ed,heterologo epitope;us and (c) a carboxy-term endoplinal, asm retiic culum retention/ret signalrieva motl ifof a membe rof the KDEL family. For certain further embodiments, the Shiga toxin effect polypeptor ideis capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directi intrng acell routiularng to the endoplasm retiic culum and/or cytosol of a cell in which the polypeptide is present inhibiti, nga ribosome function, enzymatic allinacty ivat a ingribosome causi, ng cytostas and/oris, causing cytotoxicity. In certai furthen embodimentsr the ,heterologou T-cells, epitope is a CD8+ T-cel epitope,l such as, e.g., wit hregard to a human immune system. For certai furthen embodimr ents, the heterologous, T-cel epitopel is capable of being present byed a MHC cla ssI molecul ofe a cell In. certa furtin her embodiments the, cell-targeti molecng ule of the present inventio is ncapable of one or more the follow ing:entering a cel l,inhibiting a ribosome function, causing cytostasis , causing cell death, and/or delivering the embedded or inserte heted, rologous, T-cell epitope to a MHC cla ssI molecul fore presenta tionon a cellul surface.ar -28-WO 2016/196344 PCT/US2016/034778 29 78. 78. 78. id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78"
[78] In certain embodimen ofts Embodiment Set 43, the carboxy-termi nal endoplasmic reticulum retention/re trievasignal motifl is selected from the group consisting of: KDEL, HDEF, HDEL, RDEF, RDEL, WDEL, YDEL, HEEF, HEEL, KEEL, REEL, KAEL, KCEL, KFEL, KGEL, KHEL, KLEL, KNEL, KQEL, KREL, KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL, KDEF, KKEL, HADL, HAEL, HIEL, HNEL, HTEL, KTEL, HVEL, NDEL, QDEL, REDL, RNEL, RTDL, RIEL, SDEL, IDEL, SKEL, STEL, and EDEL. 79. 79. 79. id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79"
[79] In certain embodimen ofts Embodiment Set 43, the embedded or inserte d, heterologous T-ce,ll epitope disrupts the endogenous, B-cell and/o T-cellr epitope region selected from the group of native positily oned Shiga toxin A Subunit regions consisting of: (i) 1-15 of SEQ ID NO: 1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 2b 37 of SEQ ID N():3; 27 37 of SEQ ID NO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42 48 of SEQ ID NO: x and 53-66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, or the equivale regint on in a Shiga toxin A Subuni t or derivati thereofve (ii); 94—115 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3: 179-190 of SEQ ID NO: I or SEQ ID NO 2: 179-191 of SEQ ID NO:3: 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; and 210-218 of SEQ ID NO:3: and (iii) 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO: I or SEQ ID NO:2; 254-268 of SEQ ID NO: I or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO:1 or SEQ ID NO:2, or the equivale regionnt in a Shiga toxin A Subunit or derivative thereof. 80. 80. 80. id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80"
[80] In certain furthe embodimr ents of Embodiment Set 43, the heterologo us epitope is a CD8+ T-cell epitope capable of being presente byd a MHC class 1 molecule of a cell In. certain further embodiments, the heterologous epitope in is embedded and replace an sequivalent numbe rof amino acid residues in a wild-type Shiga toxin polypeptide region such that the Shiga toxin effector polypeptide has the same total number of amino acid residues as does the wild-type Shiga toxin polypeptide regio frn om which it is derived. In certain furthe embodimr ents of any of the above, the Shiga toxin effector polypeptide is capable of exhibiting at leas t one Shiga toxin effecto functionr selected from: directin intrag cellul routingar to a cytosol of a cel inl which the polypeptide is present, inhibiting a ribosome function, enzymatically inactiva tinga ribosome, and cytotoxicity.[81] In certa embodiin ment of sEmbodime ntSet #3, the cell-targeti molengcule of the present inventi ison capable when introduce to dcell ofs exhibiting cytotoxi city that is great thaner that of a fifth cell-target moleingcule consisti ofng the cel­ l target moleing cule except for it does not comprise any carboxy-terminal, endoplasmi retic culum retention/ret signrievalal moti off the KDEL family. In certa furthein embodimr ents, the cell-target molingecule of the present invention is capable of exhibiting a cytotoxici witty hbetter optimized, cytotoxic potency such, as, e.g., 4-fold, 5-fol d,6-fold, 9-fold, or greater cytotoxici as tycompared to the fifth cell-targeti molecng ule. In certa furtherin embodiments, the cytotoxi ofcit they cell- target moling ecule of the present inventio to na population of target positive cell iss 3-fold, 4-fold, 5-fol d,6-fold, 7-fol d,8-fold 9-fold,, 10-fold or greate thanr the cytotoxici of thety fifth cell-targeti molecng ule to a second populat ionof target positive cell ass assaye byd CD50 values. 82. 82. 82. id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82"
[82] For certai embodin ment of sEmbodiment Set #3, the cell-targeti molengcule of the present invention is capabl ofe deliver ingan embedded or inserted, heterologous CD8+, T-cel epitopel to a MHC clas Is presenta tipathwaon ofy a cell for cell-surfa presentace tionof the epitope bound by a MHC clas I smolecule. 83. 83. 83. id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83"
[83] In certa embodiin ment of sEmbodiment Set #3, the cell-targeti molengcule is de-immunized due to the embedded or inserted, heterologo epitusope. In certai n further embodiments the ,cell-target molingecule is capable of exhibiting less relat iveantigeni citand/ory relat iveimmunogenic asit compay red to a reference molecule, such as, e.g., a sixt hcell-target molingecul consise ting of the cell­ target molecing ule except for it lacks one or more embedded or insert epitopesed present in the cell targeti moleng cule. 84. 84. 84. id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84"
[84] For certai furthen embodir ment of sEmbodime ntSet #3, the cell-targeting molecule of the present invention is not cytotoxic and is capabl whene introduced to cell ofs exhibiting a great subceller ular routing efficiency from an extracell ular space to a subcellular compartme ofnt an endoplasmic reticulum and/or cytosol as compared to the subcellular routing efficiency of a reference molecule, such as, e.g., the fift cellh -targeti molengcule. -30-Embodime ntSet #4 - Cell-Targeting Molecule Comprising, a Shiga Toxin Effector Polypeptide Comprisin (i)g an Embedded or Inserted, Heterologous, T-Cel lEpitope and (ii) a Disrupted, Furin-Cleava Motifge 85. 85. 85. id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85"
[85] The present inventi provideson cell-targeti molengcules eac, hcomprising (i) a binding region capable of specificall bindingy an extracel targetlular biomolecule; (ii) a Shiga toxin effector polypepti compride sing an inserted or embedded, heterologo epitusope; and (iii) a disrupted furin-cleavage motif. In certain embodiments, the cell-target molingecule of the present inventio comprin ses (i) a binding region capable of specifical bindingly an extracell targetular biomolecule; (ii) a Shiga toxin effector polypept idecomprising (a) an inserted or embedded, heterologous epitope; (b) a Shig atoxin Al fragme derivednt region having a carboxy terminus; and (c) a disrupted furin-cleavage moti atf the carboxy-term inus of the Al fragme region.nt For certa furtherin embodiment thes, Shiga toxin effector polypepti isde capabl ofe exhibiting at lea stone Shiga toxin effector function, such as, e.g., direct ingintracellul routiarng to the endoplasm retiic culum and/or cytosol of a cel inl which the polypepti isde present inhi, biting a ribosome function, enzymatical inactily vating a ribosom causinge, cytostasi and/ors, causing cytotoxici Inty. certai furthern embodiments, the heterologou T-cels, epitopel is a CD8+ T-cel epitl ope, such as, e.g., wit hregard to a human immune system. For certai furthern embodiments the ,heterologo T-celus, epitl ope is capable of being presente byd a MHC clas Is molecul ofe a cel l.In certai furthern embodiments the , cell-targeti molengcule of the present invention is capabl ofe one or more the followi ng:entering a cel l,inhibiti nga ribosome function, causing cytostasis , causing cell death, and/or delivering the embedde ord inserte heterd, ologous T-cell, epitope to a MHC clas I smolecule for presentat onion a cellular surface. For certai n further embodiments, the cell-target molingecule is capabl whene introduc toed cell s of exhibiting a cytotoxi compcity arabl or bettere than a reference molecule, such as, e.g., a second cell-targeti molngecule consisting of the cell-targeti molngecul e except for all of it sShig atoxin effector polypepti componentde compris sea wild- type Shiga toxi furin-cleavan sitege at the carboxy terminus of it sAl fragme nt region. 86. 86. 86. id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86"
[86] In certai embodin ment of sEmbodiment Set #4, the embedded or insert ed, heterologou T-ces,ll epitope disrupts the endogenous B-ce, ll and/or T-cel epitl ope region selected from the group of natively positioned Shig atoxin A Subunit regions -31-consisti of:ng (i) 1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO:1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; and 53-66 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or the equival entregion in a Shiga toxin A Subunit or derivat thereof;ive (ii) 94-115 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ IDNO:1 or SEQIDNO:2; 140-156 ofSEQIDNO:3; 179-190 of SEQ IDNO:lorSEQ IDN0:2; 179-191 of SEQ IDNO:3;204 of SEQ IDNO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; and 210-218 of SEQ ID NO:3; and (iii) 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO:1 or SEQ IDNO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO:1 or SEQ ID NO:2, or the equival entregion in a Shiga toxin A Subunit or derivat thereof.ive 87. 87. 87. id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87"
[87] In certai embodin ment of sEmbodiment Set #4, the disrupt furin-cled eavage moti comprif ses one or more mutations, relat iveto a wild-type Shiga toxin A Subunit, the mutation alteri atng least one amino acid residue in a region natively positioned at 248-251 of the A Subuni tof Shiga-like toxin 1 (SEQ ID NO: 1) or Shiga toxi (SEQn ID NO :2), or at 247-250 of the A Subunit of Shiga-li ketoxi n (SEQ ID NO:3); or the equival entregion in a Shiga toxi An Subunit or derivati ve thereof. In certai furthen embodimentsr the ,disrupt edfurin-cleava motige f compris onees or more mutations, relat iveto a wild-type Shiga toxin A Subunit, in a minimal furin cleavag sitee of the furin-cleava motige f,hi certa furthein r embodiments the ,minimal furin cleavage site is represented by the consensus amino acid sequence R/Y-x-X-R and/or R-x-x-R. 88. 88. 88. id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88"
[88] In certai embodin ment of sEmbodiment Set #4, the cell-targeti molecng ule compris aes molecular moiety located carboxy-termi to nalthe carboxy-term ofinus the Shig atoxi Aln fragment region. 89. 89. 89. id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89"
[89] In certa embodiin ment of sEmbodiment Set #4, the binding region stericall y covers the carboxy-term ofinus the Al fragme region.nt 90. 90. 90. id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90"
[90] In certa embodiin ment of sEmbodiment Set #4, the molecular moiety stericall coversy the carboxy-term ofinus the Al fragment region In. certai furthen r embodiments the ,molecular moiet comy prises the binding region. 91. 91. 91. id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91"
[91] In certa embodiin ment of sEmbodiment Set #4, the cell-targeti molengcule of the prese ntinvention comprises a binding region and/or molecular moiet locaty ed carboxy-termi to nalthe carboxy-term ofinus the Shig atoxin Al fragment region. In -32-WO 2016/196344 PCT/US2016/034778 33 certain further embodiment thes, mass of the bindin regg ion and/o molecr ular moiety is at leas 4.5t kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa, 100 kDa, or greater. 92. 92. 92. id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92"
[92] In certa embodin imen ofts Embodiment Set #4, the cell-target moleculeing compris aes bindin regiog withn a mass of at leas 4.5t kDa, 6, kDa, 9 kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-target moleingcule retains the approp ateri leve ofl the Shiga toxin biologica activl itynoted herein (e.g., cytotoxic and/ority intracell routing).ular 93. 93. 93. id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93"
[93] In certain embodiments of Embodiment Set «4, the bindin regg ion is comprise withind a relativ largely molece, ular moiet compry isi ngsuch as, e.g., a molecul moietyar with a mass of at leas 4.5t kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targetin molegcule retains the appropriat levee ofl the Shiga toxin biologic al activity noted herein. 94. 94. 94. id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94"
[94] In certain embodiments of Embodiment Set #4, the disrupted furin-cleava ge motif compris anes amino acid residue substitution in die furin-cleava motifge relat iveto a wild-type Shiga toxin A Subunit. In certain further embodiments, the substitution of the amino acid residue in the furin-cleavage motif is of an arginine residue with a non-positively charged, amino acid residue selected from the group consisting of: alanine, glycine, proline, serine, threonine aspartat, ase,paragine, glutamat glute, amine cysteine, isoleucine,, leucine, methionine, valin e, phenylalanin trypte, ophan, and tyrosine In .certa embodimein nts, the substitution of die amino acid residue in the furin-cleava motifge is of an arginine residue with a histidine. 95. 95. 95. id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95"
[95] In certa embodin imen ofts Embodiment Set #4, the cell-target moleingcule is capable when introduced to cells of exhibiting cytotoxici comparty able to the cytotoxici of tya seventh cell-targetin molecg ule consist ingof the cell-targetin g molecule except for all of its Shiga toxin effector polypeptide component(s each) comprise a wild-type Shiga toxin Al fragment and/o wild-typer Shiga toxin furin- cleava sitege at the carboxy terminu ofs its Al fragment region. In certa furthein r embodiments, the cell-targetin molegcule of the prese ntinvention is capable when introduced to cells of exhibiti ngcytotoxici thatty is in a range of from 0.1-fold, 0.5- fold, or 0.75-fold to 1.2-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fol d,or 5-fold of the cytotoxici exhitybite byd the sevent cell-h target molecing ule.[96] In certai embodin ment ofs Embodiment Set #4, the cell-target molingecule is capable when introduced to a chordate of exhibiting improved, in vivo tolerabi lity compar toed in vivo tolerabi oflit they seventh cell-targeti molengcule. 97. 97. 97. id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97"
[97] In certai embodimn ents of Embodiment Set #4, the cell-targeti molngecule is de-immunized due to the embedded or inserted, heterologous epitope. In certai n further embodiments the ,cell-targeting molecule is capable of exhibiting less relat iveantigeni citand/ory relati immve unogenici asty compared to a reference molecule such, as, e.g., an eighth cell-targeti molecng ule consisting of the cell­ target moling ecule except for it lacks one or more embedded or inserted epitopes present in the cell target molecule.ing 98. 98. 98. id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98"
[98] In certain embodiment ofs Embodiment Set #4, the cell-targeti molngecule is de-immuniz eddue to the furin-cleavage moti disf ruption. In certa furtherin embodiments, the cell-target molingecule is capabl ofe exhibiting less relati ve antigenici and/orty relat iveimmunogenicity as compared to a ninth cell-targeti ng molecule consisting of the cell-target moleingcule except for the furin- cleavage moti isf wild-type and/or all the Shiga toxi effectn polypeptor idecomponents consist of a wild-type Shiga toxin Al fragment.
Embodiment Set #5 - Cell-Targeti Moleculng Comprise ing a Shiga Toxin Effector Polypepti atde or Proxim alto an Amino-Terminus and Wherei then Shiga Toxin Effector Polypeptide Comprises an Embedded or Inserted, Heterologous. T-Cel l Epitope 99. 99. 99. id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99"
[99] The present invention provides cell-targeti molengcules each, comprising (i) a binding region capabl ofe specifical bindingly an extracell targetular biomolecule; (ii) a Shiga toxin effector polypept idecomprising an insert ored embedded, heterologous epitope; where thein Shiga toxi effen ctor polypept ideis at or proxim al to an amino-term inusof a polypeptide. In certai embodimn ents, the cell-targeti ng molecule of the present invention comprises (i) a binding region capable of specificall bindingy an extracell tarularget biomolecul (ii)e, a polypeptide component and, (iii) a Shiga toxi effectn polypor epti compride sing an inserted or embedded, heterolog epitousope; wherein the Shiga toxi effen ctor polypeptide is at or proxim toal an amino-term inusof the polypept idecomponent of the cell-targeti ng molecule. In certai furthern embodiments the ,binding region and Shig atoxi n effect polypeptor ideare physicall arrangedy or oriented within the cell-targeti ng -34-molecul suche that the binding region is not located proxim toal the amino-term inus of the Shiga toxin effect polypeor ptide In .certa furthein embodimr ents, the binding region is located within the cell-targeti molngecule more proxima to lthe carboxy­ terminus of the Shiga toxin effector polypept idethan to the amino-termin of usthe Shiga toxi effectorn polypeptide. In certai furthern embodiments the ,binding region is not located proxim toal an amino-termi ofnus the cell-target moleingcule relat ive to the Shig atoxin effect polypeor pti de.For certa furtherin embodiments the ,Shig a toxin effector polypeptide is capable of exhibiting at leas onet Shiga toxin effector function, such as, e.g., direct ingintracell routiularng to the endoplasmi retic culum and/or cytosol of a cell in which the polypept ideis present, inhibiting a ribosome function, enzymatical inactly ivat aing ribosome, causing cytosta and/orsis, causing cytotoxicit In certaiy. furthern embodiments the ,heterologous, T-cel epitopel is a CD8+ T-cell epitope, such as, e.g., with regard to a human immune syste m.For certai furthern embodiments the ,heterologous, T-cell epitope is capable of being presented by a MHC clas I smolecule of a cell. In certain further embodiments, the cell-targeti molngecul ofe the prese ntinvention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, causing cell death, and/or deliver theing embedde ord insert ed,heterologous, T-cell epitope to a MHC clas Is molecule for presentati onon a cellu larsurface. 100. 100. 100. id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100"
[100] In certain embodiment of sEmbodiment Set #5, the cell-targeti molengcule of the present invention is capable when introduce to dcel lsof exhibiting cytotoxici ty that is greater than that of a tenth cell-targeti molngecule having an amino-terminus and comprising the binding region and the Shiga toxi effectn polypeptor ideregion whic his not positioned at or proximal to the amino-terminus of the tenth cell­ target moleing cule In. certain furthe embr odiments, the cell-targeti molngecule of the present inventi ison capabl ofe exhibiting a cytotoxici wityth bett eroptimized, cytotoxic potency such, as, e.g., 4-fold, 5-fol d,6-fold, 9-fol d,or greater cytotoxici ty as compared to the tenth cell-targeti molecng ule. In certai furthern embodiments, the cytotoxic ofity the cell-targeti molngecule of the present invention to a population of target positive cell iss 3-fol d,4-fold, 5-fol d,6-fol d,7-fol d,8-fold, 9­ fold, 10-fold or greater than the cytotoxici of thety tent cell-th argeti moleculng toe a second population of target positive cel lsas assayed by CD50 values. -35-[101] For certa embodimin ents of Embodime ntSet #5, the cell-target molecing ule of the present invention is capable of delivering an embedded or insert ed, heterologo CD8+us, T-cel epitl ope to a MHC cla ssI presenta tionpathwa ofy a cell for cell-surface presenta tionof the epitope bound by a MHC cla ssI molecule. 102. 102. 102. id="p-102" id="p-102" id="p-102" id="p-102" id="p-102" id="p-102" id="p-102" id="p-102" id="p-102" id="p-102"
[102] In certai embodimen nts of Embodiment Set #5, the cell-target moleingcule is de-immunized due to the embedded or insert ed,heterolog epitopous e.In certai n further embodiments, the cell-targeti molngecule is capable of exhibiting les s relat iveantigenici and/orty relat iveimmunogenici asty compared to a reference ■ molecule, such as, e.g., an eleventh cell-targeti molngecule consisting of the cell­ target moling ecule except for it lacks one or more embedded or inserted epitopes present in the cell target moleing cule. 103. 103. 103. id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103"
[103] For certai furthern embodiments of Embodiment Set #5, the cell-target ing molecule of the present inventio is nnot cytotox andic is capable when introduced to cell ofs exhibiting a greater subcellular routing efficiency from an extracell ular space to a subcellula compar rtmen of ant endoplasm retiic culum and/or cytosol as compared to the subcellular routing efficiency of a reference molecule, such as, e.g., the tent cell-th argeti molengcule.
Embodiment Set #6 - Cell-Target Moleing cul Comprie sin a gDe-immunized Shiga Toxin Effector Polypepti Comprisde ing a Disrupted, Furin-Cleavage Motif 104. 104. 104. id="p-104" id="p-104" id="p-104" id="p-104" id="p-104" id="p-104" id="p-104" id="p-104" id="p-104" id="p-104"
[104] The present invention provides cell-target molecing ules, eac hcomprising (i) a binding region capable of specifica bindinglly an extracel targetlular biomolecule and (ii) a de-immunized, Shig atoxin effector polypept idecomprising a disrupted furin-cleava motige f.In certai embodn iments the ,cell-targeti molecng ule of the present invention comprises (i) a binding region capabl ofe specifically binding an extracell targetular biomolecule and (ii) a de-immunize Shigad, toxin effector polypeptide comprising (a) a Shiga toxin Al fragme derivednt region having a carboxy terminus, (b) a disrupt edfurin-cleavage moti atf the carboxy-term ofinus the Al fragment region, and (c) at leas onet disrupted, endogenous, B-cell and/or CD4+ T-cell epitope and/or epitope region. For certain further embodiments, the Shiga toxin effector polypepti isde capable of exhibiti ngat leas onet Shiga toxin effector function, such as, e.g., direct ingintracel lularoutirng to the endoplasmi c reticulum and/or cytosol of a cel inl which the polypept ideis present inhibit, ing a ribosome function, enzymatical inactly ivati a ribosomng causie, ng cytosta and/orsis, -36-WO 2016/196344 PCT/US2016/034778 37 causing cytotoxicity. In certai furthen embodimer nts, the cell-targeting molecule of lire prese ntinvention is capable of one or more the following: entering a cell , inhibiting a ribosome function, causing cytostasis, and/or causing cell death. For certain further embodiments, the cell-target moleculing is ecapable when introduced to ceils of exhibiting a cytotoxic compaity rable or better than a reference molecul e, such as, e.g., a second cell-target moleingcule consisting of the cell-targetin g molecule except for ail of its Shiga toxin effector polypept idecompone ntscomprise a wild-type Shiga toxin furin-cleava sitege at the carboxy terminu ofs its Al fragment region. 105. 105. 105. id="p-105" id="p-105" id="p-105" id="p-105" id="p-105" id="p-105" id="p-105" id="p-105" id="p-105" id="p-105"
[105] In certain em bodiments of Embodiment Set #6, the Shiga toxin effector polypeptide comprises a mutation, relat iveto awild-type Shiga toxin A Subunit in, the B-cel and/orl T-cell epitope region selected from the group of natively positioned Shiga toxin A Subunit regions consist ingof; 1--15 of SEQ ID NO: 1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3: 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO: 1 or SEQ ID N 0:2. 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53 66 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 of SEQ ID NO:1, SEQ ID NO :2, or SEQ ID NO:3: 141-153 of SEQ ID NO: I or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2, and 210-218 of SEQ IDNO:3; 240-260 of SEQ IDNO:3; 243-257 of SEQ IDNO:1 or SEQ ID NO:2; 254-268 of SEQ ID NO: 1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; 285-293 of SEQ ID NO:1 or SEQ ID NO:2; 4-33 of SEQ ID NO: 1 or SEQ ID NO:2; 34-78 of SEQ ID NO: 1 or SEQ ID NO:2; 77- 103 of SEQ ID NO: 1 or SEQ ID NO:2; 128-168 of SEQ ID NO: 1 or SEQ ID NO:2; 160-183 of SEQ ID NO: I or SEQ ID NO:2; 236-258 of SEQ ID NO: 1 or SEQ ID NO:2; and 274-293 of SEQ ID NO:1 or SEQ ID NO:2; or the equivale regnt ion in a Shiga toxin k Subunit or derivative thereof. In certa furthein embodimer nts, there is no disrupti onwhich is a carboxy-teiminal truncati ofon amino acid residues that overlap with part or all of at leas onet disrupted, endogenous, B-cell and/o T-cellr epitope and/or epitope region. 106. 106. 106. id="p-106" id="p-106" id="p-106" id="p-106" id="p-106" id="p-106" id="p-106" id="p-106" id="p-106" id="p-106"
[106] In certain embodiments of Embodiment Set «6, the disrupted furin-cleava ge motif compris onees or more mutations rela, tive to a wild-type Shiga toxin A Subunit, the mutation alter ingat leas onet amino acid residue in a regio natin vely positioned at 248-251 of the A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) orShiga toxin (SEQ ID NO :2), or at 247-250 of the A Subunit of Shiga-like toxin (SEQ ID NO:3); or the equivalent region in a Shig atoxin A Subunit or derivative thereof. In certain further embodiments, the disrupted furin-cleava motige f comprises one or more mutations rel, ati tove a wild-type Shiga toxin A Subunit, in a minimal furin cleavage site of the furin-cleavage moti f.In certain furthe r embodiments the ,minimal furin cleavage site is represented by the consensus amino aci dsequenc R/Ye -x-X-R and/or R-x-x-R. 107. 107. 107. id="p-107" id="p-107" id="p-107" id="p-107" id="p-107" id="p-107" id="p-107" id="p-107" id="p-107" id="p-107"
[107] In certai embodin ment of sEmbodiment Set #6, the cell-targeti molecng ule compris aes molecula moir ety locat carbed oxy-termi to thenal carboxy-terminu of s the Shig atoxi Aln fragment region. 108. 108. 108. id="p-108" id="p-108" id="p-108" id="p-108" id="p-108" id="p-108" id="p-108" id="p-108" id="p-108" id="p-108"
[108] In certa embodiin ment of sEmbodiment Set #6, the binding region sterica lly cove rsthe carboxy-term ofinus the Al fragment region. 109. 109. 109. id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109"
[109] In certai embodimn ents of Embodiment Set #6, the molecula moietr y stericall coversy the carboxy-term ofinus the Al fragment region. In certai furthen r embodiments the ,molecula moier ty comprises the binding region. 110. 110. 110. id="p-110" id="p-110" id="p-110" id="p-110" id="p-110" id="p-110" id="p-110" id="p-110" id="p-110" id="p-110"
[110] In certai embodimn ents of Embodiment Set #6, the cell-targeti molngecule of the present invention compris aes binding region and/or molecular moiety located carboxy-termi to nalthe carboxy-term ofinus the Shiga toxin Al fragment region. In certain furthe embodimr ents, the mas sof the binding region and/or molecula r moiet isy at lea st4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa ,20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa, 100 kDa, or greater. 111. 111. 111. id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111"
[111] In certai embodin ment ofs Embodiment Set #6, the cell-targeti molengcule comprises a binding region wit ha mass of at leas 4.5t kDa, 6, kDa, 9 kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or great er,as long as the cell-targeti molngecule retai thens appropri ateleve ofl the Shiga toxi n biologic actal ivit notedy herei (e.g.,n cytotoxici and/orty intracel lularouting).r 112. 112. 112. id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112"
[112] In certa embodimin ents of Embodime ntSet #6, the binding region is comprise widthi an relatively large, molecular moiety comprising such as, e.g., a molecula moier ty wit ha mass of at lea st4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa ,28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or great aser, long as the cell-target molingecule retains the appropriate leve ofl the Shiga toxi biologn ical activity noted herein. 113. 113. 113. id="p-113" id="p-113" id="p-113" id="p-113" id="p-113" id="p-113" id="p-113" id="p-113" id="p-113" id="p-113"
[113] In certa embodimin ents of Embodime ntSet #6, the disrupted furin-cleava ge mot ifcomprises an amino acid residue substitution in the furin-cleava motgeif -38-WO 2016/196344 PCT/US2016/034778 39 relat iveto a wild-type Shiga toxin A Subunit. In certain fiirthe embodimer nts, the substitution of the ammo acid residue in the furin-cleavage motif is of an arginine residue with a non-positively charged, amino acid residue select from,ed the group consisting of: alanine, glycine, proline, serine, threonine aspartat, ase,paragine, glutamat glute, amine cys, teine, isoleucine, leucine, methionine, valin e, phenylalanin tryptophan,e, and tyrosine In .certai embodimen nts, tire substitution of the amino aci dresidue in the furin-cleava motifge is of an arginine residue wit ha histidine. 114. 114. 114. id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114"
[114] In certain embodimen ofts Embodiment Set #6, the cell-target moleingcule is capable when introduced to cells of exhibiting cytotoxic׳ comparity able to the cytotoxic ofity a reference molecule, such as, e.g., a twelfth cell-targetin moleculeg consisting of the ceil-target moleingcule excep fort all of its Shiga toxin effector polypeptide component(s eac) hcomprise a wild-type Shiga toxin Al fragment and/or wild-type Shiga toxin fiirin-cleavage site at the carboxy termin ofus its Al fragment region. In certain further embodiment thes, cell-target moleingcule of the present invention is capable when introduced to cells of exhibiting cytotoxi thatcit y is in a range of from 0.1-fold, 0.5-fold, or 0.75-fold to 1.2-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold or, 5-fold of the cytotoxicity exhibite byd the twelft cell-h targeting molecule. 115. 115. 115. id="p-115" id="p-115" id="p-115" id="p-115" id="p-115" id="p-115" id="p-115" id="p-115" id="p-115" id="p-115"
[115] In certain embodimen ofts Embodiment Set #4, the cell-target moleingcule is capable when introduced to a chordate of exhibiting improved, in vivo tolerability׳ compared to in vivo tolerabili of thety twelfth cell-target molecing ule. 116. 116. 116. id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116"
[116] In certain embodimen ofts Embodiment Set #4, the cell-target moleingcule is de-immunized due to the fiirin-cleavage motif disruption. In certai fiinrthe r embodiments, the cell-targeti molengcule is capabl ofe exhibiti ngles srelati ve antigenicit and/ory relat iveimmunogenici asty compared to a reference cell- targeting molecule consisting of the cell-targeti molecng ule except for the furin- cleava motifge is wild-type and/or all the Shiga toxin effecto polypeptr ide compone ntsconsis oft a wild-type Shiga toxin Al fragment, such as, e.g., the twelfth cell-target molecing ule.WO 2016/196344 PCT/US2016/034778 40 Embodiment Set #7 --- Cell-Target Moleculeing Comprising a Carboxy-Terminal Endoplasmi Reticc ulum Retention/Retrieval Signal Motif and a De-immunize d Shiga. Toxin Effect Polypeptideor 117. 117. 117. id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117"
[117] The present invention provides cell-targeti molengcule eachs, comprising (i) a binding region capable of specifica bindinglly an extracel lulartarge biomolect ule; (ii) a de-immunized, Shiga toxin effector polypepti andde, (iii) a carboxy-termin al, endoplasmic reticulum retention/ret signarieval motif.l In certa emboin diments, the cell-target moleculeing of the prese ntinvention compris (i)es a bindin gregio n capable of specifica bindinglly an extracellular targ biomolecet ule: (ii) a de- immunized, Shiga toxin effector polypeptide comprisin atg lea stone disrupted, endogenous, B-cell and/o CD4+r T-cell epitope and/o epitoper region, and (iii) a carboxy-termi endoplasmicnal, re ticulu mretention/ret signalrieval motif of a member of the KDEL family. For certain further embodiments, the Shiga toxin effector polypeptide is capabl ofe exhibiting at least one Shiga toxin effector function, such as, e.g., directin intrg acell routingular to the endoplasm reticic ulum and/o cytosolr of a cell in which the polypept ideis present, inhibiting a ribosome function, enzymatically inactiva tinga ribosome, causing cytostasi and/ors, causing cytotoxicity. In certai furthen embodimer nts, the cell-targetin molegcule of the present invention is capable of one or more the following: entering a cel l,inhibiting a ribosome function, causing cytostas and/ois, causir ng cell death. 118. 118. 118. id="p-118" id="p-118" id="p-118" id="p-118" id="p-118" id="p-118" id="p-118" id="p-118" id="p-118" id="p-118"
[118] In certain embodimen ofts Embodiment Set #7, the carboxy-terminal endoplasmic reticulum retention/retr signaieva motifll is select fromed the group consisting of: KDEL, HDEF, HDEL, RDEF, RDEL, WDEL, YDEL, HEEF, HEEL, KEEL, REEL, KAEL, KCEL, KFEL, KGEL, KHEL, KLEL, KNEL, KQEL, KREL, KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL, KDEF, KKEL, HADL, HAEL, HIEL, HNEL, HTEL, KTEL, HVEL, NDEL, QDEL, REDE, RNEL, RTDL, RIEL, SDEL, TDEL, SKEL, STEL, and EDEL. 119. 119. 119. id="p-119" id="p-119" id="p-119" id="p-119" id="p-119" id="p-119" id="p-119" id="p-119" id="p-119" id="p-119"
[119] In certain embodiments of Embodiment Set #7, the Shiga toxin effector polypeptide comprises a mutation, relat iveto a wild-type Shiga toxin A Subunit, in tiie B-cell and/or T-cell epitope region selected from the group of natively positioned Shiga toxin A Subunit regions consist ingof: 1-15 of SEQ ID NO: 1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO: 1 or SEQ ID NO:2: 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3: 53-66 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 of SEQ IDNO:1, SEQ IDNO:2, or SEQ IDNO:3; 141-153 ofSEQ IDNO:1 or SEQ IDNO:2; 140-156 of SEQ IDN0:3; 179-190 ofSEQ ID NO:1 orSEQIDNO:2; 179-191 of SEQ ID NO:3; 204 ofSEQ ID NO:3; 205 ofSEQ ID NO:1 or SEQ ID NO:2, and 210 218 ofSEQ ID NO:3; 240-260 ofSEQ ID NO:3; 243-257 ofSEQ ID NO:1 or SEQ ID NO:2; 254-268 ofSEQ ID NO:1 or SEQ ID NO:2; 262-278 ofSEQ ID NO:3; 281-297 ofSEQ IDNO:3; 285-293 ofSEQ ID NO:1 or SEQ IDN0:2; 4-33 ofSEQ ID NO:1 or SEQ ID NO:2; 34-78 ofSEQ ID NO:1 or SEQ ID NO:2; 77­ 103 ofSEQ IDNO:1 or SEQ IDN0:2; 128-168 ofSEQ IDNO:1 or SEQ IDNO:2; 160-183 ofSEQ ID NO:1 or SEQ ID NO:2; 236-258 ofSEQ ID NO:1 or SEQ ID NO:2; and 274-293 of SEQ ID NO:1 or SEQ ID NO:2; or the equival entregion in a Shiga toxin A Subunit or derivative thereof. In certai furthen embodimentsr there, is no disruption whic his a carboxy-ter mitruncnal ati ofon amino acid residues that overlap wit hpart or all of at leas onet disrupted, endogenous, B-cel and/orl T-cell epitope and/or epitope region. 120. 120. 120. id="p-120" id="p-120" id="p-120" id="p-120" id="p-120" id="p-120" id="p-120" id="p-120" id="p-120" id="p-120"
[120] In certai embodimn ents of Embodime ntSet #7, the cell-targeti molngecule of the present inventi ison capable when introduced to cell ofs exhibiting cytotoxic ity that is great thaner that of a thirteenth cell-targeti molngecule consisti ofng the cell­ targeti moleng cule except for it does not compri seany carboxy-terminal , endoplasm reticic ulum retention/ret siriegnalval mot ifof the KDEL famil y.In certai furthern embodiments the ,cell-targeti molngecule of the present inventio is n capable of exhibiting a cytotoxi witcity hbetter optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fol d,6-fol d,9-fol d,or greater cytotoxic asity compared to the thirteenth cell-targeti molengcule. In certa furthein embodimr ents, the cytotoxici ty of the cell-target molecing ule of the present invention to a population of target positive cell iss 3-fol d,4-fold, 5-fol d,6-fold, 7-fold, 8-fol d,9-fold, 10-fold or greater than the cytotoxici of thety thirtee celnthl-targeti molengcule to a second population of target positive cell ass assayed by CDs0 values. 121. 121. 121. id="p-121" id="p-121" id="p-121" id="p-121" id="p-121" id="p-121" id="p-121" id="p-121" id="p-121" id="p-121"
[121] For certai furthern embodiment ofs Embodiment Set #7, the cell-target ing molecule of the present inventio is nnot cytotoxi andc is capabl whene introduc toed cell ofs exhibiting a great subceller ular routing efficiency from an extracell ular spac toe a subcellula compar rtmen of ant endoplasmi retic culum and/or cytosol as compared to the subcellular routing efficiency of a reference molecule, such as, e.g., the thirteent cell-th argeti molengcule. -41-Embodimen Sett #8 - Cell-Targeti Moleculng Comprie sing a De-immunized Shiga Toxin Effector Polypeptide at or Proxima tol an Amino-Terminus of the Cell Targeti ng Molecule 122. 122. 122. id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122"
[122] The present inventio providesn cell-targe moltingecul es,each comprisi ng(i) a binding region capable of specificall bindiy ng an extracel targetlular biomolecule, (ii) a de-immunized, Shig atoxin effector polypepti de;wherei then Shiga toxi effen ctor polypeptide is at or proxim toal an amino-terminus. In certa embodimin ents, the cel­ l targeti molecng ule of the present inventi oncompris (i)es a binding region capable of specificall bindingy an extracel targetlular biomolecule; (ii) polypepti componentde and; (iii) a de-immunize Shigad, toxi effen ctor polypepti compride sin atg least one disrupted, ■ endogenous, B-cell and/or CD4+ T-cel epitol pe and/or epitope region; wherei then Shig atoxi effen ctor polypepti isde at or proximal to an amino-terminus of the polypepti compde onent of the cell-targe moltingecule. In certai furtn her embodiment s, the binding region and Shig atoxin effector polypept ideare physica llyarranged or orient wited hin the cell-target molecing ule such that the binding region is not located proxim alto the amino-term inusof the Shiga toxi effen ctor polypeptide. In certa in further embodiment thes, binding region is locate witd hin the cell-target molingecule more proximal to the carboxy-termi ofnus the Shiga toxi effen ctor polypepti thande to the amino-terminus of the Shig atoxin effector polypeptide. In certai furtn her embodiment thes, binding region is not locat proximaed tol an amino-term inusof the cell-target molecing ule relative to the Shig atoxi effen ctor polypeptide. For certain further embodiment thes, Shig atoxin effector polypept ideis capabl ofe exhibiti ngat least one Shig atoxin effecto functir on, such as, e.g., directing intracell routiularng to the endoplasmi reticuc lum and/or cytosol of a cel inl whic hthe polypepti isde present, inhibiting a ribosom functie on, enzymatical inactily vating a ribosome causi, ng cytostasis, and/or causing cytotoxici Inty. certai furthern embodiments, the cell­ targeti molecng ule of the present invention is capabl ofe one or more the following: enteri ang cell, inhibiting a ribosome function, causing cytostasi and/ors, causing cel l death. 123. 123. 123. id="p-123" id="p-123" id="p-123" id="p-123" id="p-123" id="p-123" id="p-123" id="p-123" id="p-123" id="p-123"
[123] In certa embodiin ment ofs Embodiment Set #8, the Shig atoxi effectorn polypeptide comprises a mutation, relat iveto a wild-type Shig atoxi An Subunit, in the B-cell and/or T-cell epitope region select fromed the group of natively positioned Shiga toxi An Subunit regions consist ingof: 1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID -42-WO 2016/196344 PCT/US2016/034778 43 NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO: 1, SEQ ID \O:2. or SEQ ID NO:3; 94-115 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3: 141-153 of SEQ ID NO: I or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2, and 210-218 of SEQ IDNO:3; 240-260 of SEQ IDNO:3; 243-257 of SEQ IDNO:1 or SEQ ID NO:2; 254-268 of SEQ ID NO: 1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; 285-293 of SEQ ID NO: I or SEQ ID NO:2; 4-33 of SEQ ID NO: I or SEQ ID NO:2; 34 78 of SEQ ID NO: 1 or SEQ ID NO:2; 77- 103 of SEQ ID NO: 1 or SEQ ID NO:2; 128-168 of SEQ ID NO: 1 or SEQ ID NO:2; 160-183 of SEQ ID NO: 1 or SEQ ID NO:2; 236-258 of SEQ ID NO: 1 or SEQ ID NO:2; and 274-293 of SEQ ID NO: I or SEQ ID NO:2; ortho equivale regnt ion in a Shiga toxin A Subunit or derivative thereof. In certa furthein embodimer nts, there is no disruption which is a carboxy-teiminal truncation of amino acid residues that overlap with part or all of at leas onet disrupted, endogenous, B-cell and/o T-cellr epitope and/or epitope region. [12,4] In certain embodimen ofts Embodiment Set #8, the cell-target moleingcule of the prese ntinvention is capable when introduced to cells of exhibiting cytotoxic ity' that is great thaner that of a fourteenth cell-targeti molengcule having an amino- termin andus comprisin theg binding regio andn the Shiga toxin effector polypeptide region which is not positioned at or proximal to the amino-termi ofnus the fourteenth cell-target moleingcule In. certain further embodiments, the cell-target moleculeing of the prese ntinvention is capabl ofe exhibiting a cytotoxici witty hbette optimizr ed, cytotoxic potenc suchy, as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or great cytotoxicer ity as compared to the fourteenth cell-target moleingcule In. certain further embodiments, the cytotoxi ofcit they cell-target moleingcule of the present invention to a population of targe positt ive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or great thaner the cytotoxici of thety fourtee nthcell-targeting molecule to a second population of targ positet ive cel lsas assayed by CD50 values. 125. 125. 125. id="p-125" id="p-125" id="p-125" id="p-125" id="p-125" id="p-125" id="p-125" id="p-125" id="p-125" id="p-125"
[125] For certain furthe embodimer nts of Embodiment Set #8, the cell-targeting molecule of the prese ntinvention is not cytotoxic and is capable when introduce to d cel lsof exhibiting a great subceller ular routin effg icienc fromy an extracellula r space to a subcellular compartment of an endoplasmic reticulum and/o cytosr asolcompared to the subcellular routing efficiency of a reference molecule, such as, e.g., the fourtee nthcell-target moleingcule.
Embodiment Set #9 - Cell-Targeti Moleng cule Comprising a Carboxy-Termin al Endoplasm Retiic culum Retention/Retrieval Signa Motil andf a Shiga Toxin Effector Polypepti Compride sing a Disrupted, Furin-Cleav ageMotif 126. 126. 126. id="p-126" id="p-126" id="p-126" id="p-126" id="p-126" id="p-126" id="p-126" id="p-126" id="p-126" id="p-126"
[126] The present inventio provin des cell-targeti molengcules each, comprising (i) a binding region capabl ofe specifical bindily ng an extracel targetlular biomolecule; (ii) a Shiga toxi effectn polypeptior compride sing a disrupt edfurin-cleava motige f; and (iii) a carboxy-termi endoplnal asm reticicul umretention/retri signevalal moti f.
The present invention provide cells -targeti molengcules eac, hcomprising (i) a binding region capable of specifical bindingly an extracel targetlular biomolecule; (ii) a Shiga toxin effector polypept idecomprising a disrupt edfurin-cleava motifge ; and (iii) a carboxy-term endoplinal, asm retiic culum retention/retr signalieval mot if of a membe rof the KDEL famil y.For certai furthen embr odiments the ,Shiga toxin effector polypepti isde capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directi intrang cel lularoutirng to the endoplasm retiic culum and/or cytosol of a cel inl which the polypept ideis present inhi, biting a ribosome function, enzymatical inactly ivati a ribosomeng causi, ng cytostas and/oris, causing cytotoxici Inty. certai furthen embr odiments the ,cell-targeti molngecule of the present inventio is ncapable of one or more the followi ng:entering a cell, inhibiting a ribosome function, causing cytostasi and/ors, causing cell death. For certai n furthe embodimr ents, the cell-targeti molengcule is capable when introduced to cell s of exhibiting a cytotoxi compcity arabl or ebett erthan a reference molecule, such as, e.g., a second cell-targeti molngecul consie sti ofng the cell-targeti molngecul e except for all of its Shiga toxin effect polypeptor idecomponents comprise a wil­d type Shiga toxin furin-cleava sitge eat the carboxy termin ofus it sAl fragme nt region. 127. 127. 127. id="p-127" id="p-127" id="p-127" id="p-127" id="p-127" id="p-127" id="p-127" id="p-127" id="p-127" id="p-127"
[127] In certai embodin ment ofs Embodiment Set #9, the disrupt edfurin-cleava ge mot ifcomprises one or more mutations, relat iveto a wild-type Shiga toxin A Subunit, the mutati onalteri atng leas onet amino aci dresidue in a region natively positioned at 248-251 of the A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) or Shiga toxi (SEQn ID NO:2), or at 247-250 of the A Subunit of Shiga-li ketoxi n (SEQ ID NO:3); or the equivale regionnt in a Shiga toxin A Subuni tor derivat ive -44-thereof. In certa furthein embodimr ents, the disrupted furin-cleavage mot if compris onees or more mutations relat, iveto a wild-type Shiga toxi An Subunit, in a minimal furi ncleavage site of the furin-cleava motige f.In certai furthen r embodiments the ,minimal furin cleavag sitee is represented by the consensu amis no aci dsequence R/Y-x-x-R and/or R-x-x-R. 128. 128. 128. id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128"
[128] In certai embodin ment of sEmbodiment Set #9, the cell-targeti molngecule compris aes molecul moiar ety locat carboxy-termied to thenal carboxy-terminu of s the Shiga toxi Aln fragme region.nt 129. 129. 129. id="p-129" id="p-129" id="p-129" id="p-129" id="p-129" id="p-129" id="p-129" id="p-129" id="p-129" id="p-129"
[129] In certai embodin ment of sEmbodime ntSet #9, the binding region stericall y covers the carboxy-term ofinus the Al fragment region. 130. 130. 130. id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130"
[130] In certa embodiin ment of sEmbodime ntSet #9, the molecula moier ty stericall coversy the carboxy-term ofinus the Al fragment region. In certai furthern embodiments, the molecul moietar comprisy thees binding region, 131. 131. 131. id="p-131" id="p-131" id="p-131" id="p-131" id="p-131" id="p-131" id="p-131" id="p-131" id="p-131" id="p-131"
[131] In certai embodin ment of sEmbodiment Set #9, the cell-targeti molecng ule of the present invention compris aes binding region and/or molecula moietr locaty ed carboxy-termi to nalthe carboxy-term ofinus the Shiga toxi Aln fragme regint on. In certain further embodiments, the mas sof the binding region and/or molecular moiet isy at leas 4.5t kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa, 100 kDa, or greater. 132. 132. 132. id="p-132" id="p-132" id="p-132" id="p-132" id="p-132" id="p-132" id="p-132" id="p-132" id="p-132" id="p-132"
[132] In certai embodin ment of sEmbodiment Set #9, the cell-targeting molecule compris aes binding region wit ha mass of at leas 4.5t kDa, 6, kDa, 9 kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or great aser, long as the cell-targeti molengcule retai thens appropriat levee ofl the Shiga toxin biological activity noted herei (e.g.,n cytotoxic and/ority intracel lularoutirng). 133. 133. 133. id="p-133" id="p-133" id="p-133" id="p-133" id="p-133" id="p-133" id="p-133" id="p-133" id="p-133" id="p-133"
[133] In certa embodiin ment ofs Embodiment Set #9, the binding region is comprised withi an relatively large, molecul moiar ety comprising such as, e.g., a molecula moier ty with a mas sof at leas 4.5t kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or great er,as long as the cell-targeti molngecule retains the appropriat levee ofl the Shiga toxi bioln ogic al activ itynoted herein. 134. 134. 134. id="p-134" id="p-134" id="p-134" id="p-134" id="p-134" id="p-134" id="p-134" id="p-134" id="p-134" id="p-134"
[134] In certa embodimin ents of Embodime ntSet #9, the disrupt edfurin-cleavage mot ifcomprises an amino acid residue substitution in the furin-cleava motifge relat toive a wild-type Shiga toxin A Subunit. In certai furthern embodiments the , substituti ofon the amino acid residue in the furin-cleavage mot ifis of an arginine -45-WO 2016/196344 PCT/US2016/034778 46 residue with a non-positively charged, amino acid residue selected from die group consisting of: alanine, glycine, proline, serine, threonine, aspartat ase,paragine, glutamat glute, amine cys, teine, isoleucine, leucine, methionine, valin e, phenylalanin tryptophae, andn, tyrosine In .certai embodimen nts, the substitution of the amino acid residue in the furin-cleava motifge is of an arginine residue with a histidine. 135. 135. 135. id="p-135" id="p-135" id="p-135" id="p-135" id="p-135" id="p-135" id="p-135" id="p-135" id="p-135" id="p-135"
[135] In certain embodimen ofts Embodiment Set #9, the cell-target moleingcule of die prese ntinvention is capable when introduced to cells of exhibiting cytotoxicity dial is great thaner that of a fifteenth cell-target moleculeing consist ingof the cell- targeting molecule excep fort it does not compris anye carboxy-termina l, endoplasmic reticulum retention/retr signalieval motif of the KDEL family. In certain further embodiments, the ceil-target moleculing ofe the present invention is capable of exhibiting a cytotoxicity with better optimized, cytotoxic potenc suchy, as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or great cytoter oxici as tycompared to the fifteenth cell-targeti molengcule In. certa furtherin embodiment thes, cytotoxici of ty die cell-targeti molecng ule of die present invention to a population of targe positivet cells is 3-fold, 4-fol d,5-fold, 6-fol d,7-fold, 8-fold, 9-fold, 10-fold or great thaner the cytotoxici of thety fifteenth cell-target moleculeing to a. second population of targe positt ive cells as assaye byd CD50 values. 136. 136. 136. id="p-136" id="p-136" id="p-136" id="p-136" id="p-136" id="p-136" id="p-136" id="p-136" id="p-136" id="p-136"
[136] In certain embodimen ofts Embodiment Set #9, the cell-target moleingcule is capabl e when introduced to a chordate of exhibiting improved, in vivo tolerabili ty' compared to in vivo tolerabili of tya sixteenth cell-targetin molecg ule consist ingof die cell-targeti molecng ule except for all of its Shiga toxin effector polypept ide component(s) eac hcompri sea wild-type Shiga toxin Al fragment and/o wild-r type Shiga, toxin furin-cleavage sit eat the carboxy terminus of its Al fragment region. 137. 137. 137. id="p-137" id="p-137" id="p-137" id="p-137" id="p-137" id="p-137" id="p-137" id="p-137" id="p-137" id="p-137"
[137] In certain embodimen ofts Embodiment Set #9, the cell-target moleingcule is de-immuniz eddue to the funn-cleavage motif disruption. In certai furthen r embodiments, the cell-targeti molengcule is capabl ofe exhibiti ngles srelati ve antigeni cityand/or relat iveimmunogenic7 asity compared to a refere ncecell- targeti moleng cule consisting of the cell-targeti molecng ule except for the furin- cleava motifge is wild-type and/o allr the Shiga toxin effector polypept ide compone ntsconsis oft a wild-type Shiga toxin Al fragment, such as, e.g., the sixteenth cell-targeti molengcule.[138] For certa furtherin embodiment ofs Embodiment Set #9, the cell-targeti ng molecul ofe the present inventi ison not cytotoxi andc is capable when introduced to cell ofs exhibiting a greater subcellular routing efficiency from an extracell ular spac toe a subcellula compartr ment of an endoplasmi retic culum and/or cytosol as compared to the subcellula routr ing efficiency of a reference molecule, such as, e.g., the fifteent cellh -targeti molengcule.
Embodiment Set #10 - Cell-Target Moleculeing Comprising a Furin-Cleavage Resistan Shigt aToxin Effector Polypepti atde or Proxim alto an Amino-Terminus of the Cel lTarget ingMolecule . [139] The prese ntinvention provide cell-ts argeti molecng ules, eac hcomprising (i) a binding region capable of specifical bindly ing an extracell targetular biomolecule and (ii) a Shiga toxin effector polypeptide comprising a disrupt edfurin-cleava ge moti atf the carboxy-termi ofnus its Shiga toxi Aln fragment regio n;where thein amino-term inusof the Shig atoxin effector polypeptide is at and/or proxim toal an amino-term inusof a polypept idecomponent of the cell-target moleingcule. In certai embodimn ents, the cell-targeti molngecule of the present inventio comprin ses (i) a binding region capable of specifica bindlly ing an extracellu targetlar biomolecul (ii)e, a Shiga toxin effector polypept idehaving an amino-termin andus a Shiga toxin Al fragme derivednt region having a carboxy terminus, and (iii) a disrupted furin-cleava motge ifat the carboxy-term ofinus the Al fragme region;nt wherein the binding region is not locat proximed toal the amino-termi ofnus the cell­ target moling ecul relate iveto the Shiga toxi effen ctor polypepti de.In certai furthern embodiments the ,binding region and Shiga toxin effector polypept ideare physically arrang ored oriented withi then cell-targeti molecng ule such that the binding region is not locate proximd toal the amino-terminus of the Shig atoxin effector polypepti de.In certa furthein embodimr ents, the binding region is locate withid n the cell-targeti molngecule more proxim toal the carboxy-termi ofnus the Shiga toxi effectorn polypept idethan to the amino-termin of usthe Shig atoxin effector polypeptide. In certai furthen embr odiments the ,binding region is not located proxim toal an amino-termi ofnus the cell-targeti molengcule relat toive tire Shig a toxi effen ctor polypeptid Fore. certai furtn her embodiments, the Shiga toxin effector polypeptide is capable of exhibiting at lea stone Shiga toxi effen ctor function, such as, e.g., direct ingintracel lularoutring to the endoplasm retiic culum -47-and/or cytosol of a cell in which the polypept ideis present inhi, biting a ribosome function, enzymatical inactly ivati a ribosong me, causing cytosta and/orsis, causing cytotoxi citIn certaiy. furthen embodir ment thes, cell-targeti molngecule of the present inventi ison capable of one or more the follow ing:enteri ang cell, inhibiti ng a ribosome function, causing cytostasis, and/or causing cel death.l For certai n further embodiments, the cell-targeti molngecul is ecapable when introduc toed cell s of exhibiting a cytotoxici compty arab orle better than a reference molecul suche, as, e.g., a seventeenth cell-targeti molngecul consise ti ofng the cell-targeti molengcule except for all of its Shiga toxi effecn tor polypept idecomponent comps rise a wild­ type Shiga toxi furin-cln eavage site at the carboxy terminus of its Al fragme nt region. 140. 140. 140. id="p-140" id="p-140" id="p-140" id="p-140" id="p-140" id="p-140" id="p-140" id="p-140" id="p-140" id="p-140"
[140] In certai embodimn ents of Embodiment Set #10, the disrupted furin-cleavage moti comf prises one or more mutations, relati tove a wild-type Shiga toxi An Subunit, the mutati onalterin at gleast one amino acid residue in a region natively positioned at 248-251 of the A Subunit of Shiga-li ketoxi 1n (SEQ ID NO:1) or Shiga toxi (SEQn ID NO:2), or at 247-250 of the A Subuni tof Shiga-like toxi n (SEQ ID NO:3); or the equivale regionnt in a Shiga toxi An Subunit or derivat ive thereof. In certain furthe embodimr ents, the disrupted furin- cleavag mote if comprises one or more mutations relative, to a wild-type Shiga toxi An Subunit, in a minimal furin cleava sitege of the furin-cleava motige f.In certai furtn her embodiments, the minimal furin cleavage sit eis represent byed the consensus amino acid sequenc R/Ye -x-x-R and/or R-x-x-R. 141. 141. 141. id="p-141" id="p-141" id="p-141" id="p-141" id="p-141" id="p-141" id="p-141" id="p-141" id="p-141" id="p-141"
[141] In certai embodimn ents of Embodiment Set #10, the cell-targeting molecule comprise a smolecula moietr locay ted carboxy-termi to nalthe carboxy-termi ofnus the Shig atoxin Al fragme region.nt 142. 142. 142. id="p-142" id="p-142" id="p-142" id="p-142" id="p-142" id="p-142" id="p-142" id="p-142" id="p-142" id="p-142"
[142] In certai embodimn ents of Embodiment Set #10, the binding region sterically cove rsthe carboxy-termi ofnus the Al fragme region.nt 143. 143. 143. id="p-143" id="p-143" id="p-143" id="p-143" id="p-143" id="p-143" id="p-143" id="p-143" id="p-143" id="p-143"
[143] In certai embodimn ents of Embodiment Set #10, the molecula moietr y stericall coversy the carboxy-terminu of thes Al fragme region.nt In certai furthen r embodiments, the molecul moietar comprisesy the binding region. 144. 144. 144. id="p-144" id="p-144" id="p-144" id="p-144" id="p-144" id="p-144" id="p-144" id="p-144" id="p-144" id="p-144"
[144] In certa emboin diments of Embodiment Set #10, the cell-targeti molngecule of the present invention comprises a binding region and/or molecul moietar locay ted carboxy-termi to nalthe carboxy-termi ofnus the Shiga toxi Aln fragme region.nt In certain further embodiments, the mass of the binding region and/or molecular -48-moiet isy at leas 4.5t kDa ,6, kDa ,9 kDa, 12 kDa ,15 kDa ,20 kDa, 25 kDa ,28 kDa, kDa, 41 kDa, 50 kDa, 100 kDa, or greater , . 145. 145. 145. id="p-145" id="p-145" id="p-145" id="p-145" id="p-145" id="p-145" id="p-145" id="p-145" id="p-145" id="p-145"
[145] In certain embodiment ofs Embodime ntSet #10, the cell-target molecing ule compris aes binding region wit ha mas sof at leas 4.5t kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or great er,as long as the cell-targeti molngecul retaie thens appropriat levele of the Shig atoxin biological activi notedty herei (e.g.,n cytotoxici and/orty intracel lulrouting).ar 146. 146. 146. id="p-146" id="p-146" id="p-146" id="p-146" id="p-146" id="p-146" id="p-146" id="p-146" id="p-146" id="p-146"
[146] In certai embodin ment of sEmbodime ntSet #10, the binding region is comprised within a relativel laryge, molecul moietar compriy sing such as, e.g.,a molecula moietr wity ha mas sof at leas 4.5t kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or great er,as long as the cell-targeti molngecule retai thens appropriat levee ofl the Shiga toxin biological activi notedty herein. 147. 147. 147. id="p-147" id="p-147" id="p-147" id="p-147" id="p-147" id="p-147" id="p-147" id="p-147" id="p-147" id="p-147"
[147] In certain embodiments of Embodiment Set #10, the disrupted furin-cleava ge moti comprisf anes amino acid residue substitution in the furin-cleava motige f relat iveto a wild-type Shiga toxin A Subunit. In certai furthern embodiments, the substitution of the amino acid residue in the furin-cleava motge ifis of an arginine residue wit ha non-positively charged, amino acid residue selected from the group consisti of:ng alanine, glycine, proline, serine, threoni aspartatne, asparagie, ne, glutamat glutamie, ne,cysteine, isoleucine, leucine, methioni ne,valine, phenylalanine, tryptophan, and tyrosine. In certai embodimn ents, the substitution of the amino acid residue in the furin-cleavage moti isf of an arginine residue wit ha histidine. 148. 148. 148. id="p-148" id="p-148" id="p-148" id="p-148" id="p-148" id="p-148" id="p-148" id="p-148" id="p-148" id="p-148"
[148] In certai embodimen nts of Embodiment Set #10, the cell-targeti molengcule of the present inventio is ncapable when introduce to dcell ofs exhibiting cytotoxici thatty is greater than that of an eighteenth cell-targeti molngecule having an amino-termi andnus comprising the binding region and the Shiga toxin effector polypept ideregion which is not positioned at or proxim toal the amino-termi ofnus the eightee nthcell-targeting molecule In. certai furthern embodiments the ,cell- target moling ecule of the present inventio is ncapable of exhibiting a cytotoxicity wit hbett eroptimized, cytotoxi potencyc such, as, e.g., 4-fold, 5-fol d,6-fold, 9-fol d, or greate cytor toxi ascit comparedy to the eighteenth cell-targeti molengcule In. certai furthern embodiments, the cytotoxic ofity the cell-targeti molngecul ofe the present invention to a population of target positive cell iss 3-fold, 4-fold, 5-fold, 6- -49-fold, 7-fold 8-fol, d,9-fold, 10-fol dor great thaner the cytotoxici of tythe eightee nth cell-targeti molengcule to a second populat ionof target positive cell ass assayed by CD50 values. • 149. 149. 149. id="p-149" id="p-149" id="p-149" id="p-149" id="p-149" id="p-149" id="p-149" id="p-149" id="p-149" id="p-149"
[149] In certai embodimn ents of Embodiment Set #10, the cell-target molingecule is capable when introduce to da chorda ofte exhibiting improved, in vivo tolerabi lity compared to in vivo tolerabi ofli aty nineteenth cell-targeti molngecule consisti ofng the cell-targeti molengcule except for all of its Shiga toxin effect polyor pept ide component(s) each compri sea wild-type Shiga toxin Al fragment and/or wild-type Shiga toxin furin-cleava sitge eat the carboxy terminus of its Al fragme region.nt 150. 150. 150. id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150"
[150] In certai embodin ment of sEmbodiment Set #10, the cell-targeti molengcule is de-immunized due to the furin-cleava motige disf ruption. In certain further embodiments the ,cell-targeti molngecul is ecapable of exhibiti ngles rels ati ve antigenici and/orty relat iveimmunogenic asity compared to a reference cell­ targeti moleng cule consisting of the cell-target molingecule except for the furin- cleavag mote ifis wild-type and/or all the Shiga toxin effector polypepti de components consis oft a wild-type Shiga toxi Aln fragment, such as, e.g., the nineteenth cell-targeti molengcule. 151. 151. 151. id="p-151" id="p-151" id="p-151" id="p-151" id="p-151" id="p-151" id="p-151" id="p-151" id="p-151" id="p-151"
[151] For certai furthern embodiments of Embodiment Set #10, the cell-targeti ng molecul ofe the present inventio is nnot cytotoxi andc is capable when introduced to cell ofs exhibiting a greater subcellular routing efficiency from an extracell ular space to a subcellular compartm ofent an endoplasm retiic culum and/or cytoso as l compared to the subcellular routing efficiency of a reference molecule, such as, e.g., the nineteenth cell-targeti molengcule.
Embodime ntSet #11- Cell-Target Moleing cule Comprising a Carboxy-Termi nal Endoplasmi Retic culum Retention/Retrieval Signa Motifl and Shig aToxin Effector Polypepti atde or Proxim toal an Amino-Terminus of the Cel lTarget ingMolecul e 152. 152. 152. id="p-152" id="p-152" id="p-152" id="p-152" id="p-152" id="p-152" id="p-152" id="p-152" id="p-152" id="p-152"
[152] The present inventio providen cells -targeti molengcules each, comprising (i) a binding region capable of specifica bindlly ing an extracell targetular• biomolecule, (ii) a carboxy-termi endoplnal, asm reticicul umretention/ret signrievaal motil f,and (iii) a Shiga toxi effectn polypeor ptid wheree; thein amino-terminus of the Shiga toxin effector polypept ideis at and/or proxim toal an amino-termi ofnus a polypept idecomponent of the cell-targeti molengcule In. certain embodiments, the cell-targeti molngecule of the present invention comprises a (i) binding region -50-capabl ofe specifical bindingly an extracel targetlular biomolecule, (ii) a carboxy­ termina endopll, asm retiic culum retention/ret signalrieval moti off a membe rof the KDEL family, (iii) a polypept idecomponent, and (iv) a Shiga toxin effector polypeptide; where thein amino-term inusof the Shiga toxi effen ctor polypepti isde at and/or proxim toal an amino-termi ofnus a polypept idecomponent of the cell­ target molecing ule. In certa furthein embodimr ents, the binding region and Shiga toxin effector polypeptide are physicall arrangedy or oriented withi then cell­ target moling ecule such that the binding region is not locat proximaed tol the amino­ terminus of the Shiga toxi effectorn polypeptide. In certai furthern embodiments, the binding region is locat wited hin the cell-targeti molngecule more proxim toal the carboxy-termi ofnus the Shiga toxi effen ctor polypepti thande to the amino-termi nus of the Shiga toxin effector polypeptide In .certai furthern embodiments the ,binding region is not located proxim toal an amino-termin of usthe cell-target moleingcule relati tove the Shiga toxin effector polypeptide. 153. 153. 153. id="p-153" id="p-153" id="p-153" id="p-153" id="p-153" id="p-153" id="p-153" id="p-153" id="p-153" id="p-153"
[153] For certai furthern embodiments the, Shiga toxin effector polypept ideis capable of exhibiting at leas onet Shiga toxi effen ctor function, such as, e.g., directi intngracell routularing to the endoplasmi retic culum and/or cytos ofol a cell in whic hthe polypeptide is present, inhibiting a ribosom functie on, enzymatic ally inactivati a ribong some caus, ing cytosta and/orsis, causing cytotoxi citIn certaiy. n furthe embr odiments the ,cell-target molingecule of the present inventi ison capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, and/or causing cel death.l 154. 154. 154. id="p-154" id="p-154" id="p-154" id="p-154" id="p-154" id="p-154" id="p-154" id="p-154" id="p-154" id="p-154"
[154] In certai embodin ment of sEmbodiment Set #11, the carboxy-termi nal endoplasm retiic culum retention/retrie signaval motil isf selected from the group consisting of: KDEL, HDEF, HDEL, RDEF, RDEL, WDEL, YDEL, HEEF, HEEL, KEEL, REEL, KAEL, KCEL, KFEL, KGEL, KHEL, KLEL, KNEL, KQEL, KREL, KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL, KDEF, KKEL, HADL, HAEL, HIEL, HNEL, HTEL, KTEL, HVEL, NDEL, QDEL, REDL, RNEL, RTDL, RTEL, SDEL, IDEL, SKEL, STEL, and EDEL. 155. 155. 155. id="p-155" id="p-155" id="p-155" id="p-155" id="p-155" id="p-155" id="p-155" id="p-155" id="p-155" id="p-155"
[155] In certai embodimn ents of Embodiment Set #11, the cell-target molingecule of the present invention is capab lewhen introduced to cells of exhibitin g cytotoxici thatty is greater than that of a twentieth cell-targeti molngecul havinge an amino-terminus and comprising the binding region and the Shig atoxin effect or polypept ideregion whic his not positioned at or proxima to lthe amino-term inusof -51-the twentieth cell-targeti molngecule and/or great thaner that of a twenty-f cellirst­ target moling ecule consisting of the cell-targeti molngecule except for it does not comprise any carboxy-termi endoplnal, asm retiic culum retention/retriev signal al moti off the KDEL family. In certai furthen embr odiments, the twenti ethcel l- targeti molng ecule does not comprise any carboxy-terminal endoplasm, retiic culum retention/retri signaleval mot ifof the KDEL famil y.In certa furtherin embodiments , the cell-target molingecule of the prese ntinvention is capable of exhibiting a cytotoxici witty hbetter optimized, cytotoxi potency,c such as, e.g., 4-fol d,5-fold, 6­ fold, 9-fol d,or greater cytotoxici as tycompared to a reference molecule, such as, e.g., the twentieth and/or twenty-f cell-tirst argeti molengcule Ins. certai furthern . embodiments, the cytotoxici of thety cell-target molingecule of the present invention to a populat ionof target positive cell iss 3-fol d,4-fold, 5-fold, 6-fold, 7-fol d,8-fol d, 9-fold, 10-fol ord greate thanr the cytotoxici of thety twenti ethand/or twenty-f irst cell-targeti molengcule to sa second population of target positive cell ass assaye byd CD50 values. 156. 156. 156. id="p-156" id="p-156" id="p-156" id="p-156" id="p-156" id="p-156" id="p-156" id="p-156" id="p-156" id="p-156"
[156] For certai furthern embodiment of sEmbodiment Set #11, the cell-targeting . molecule of the present inventio is nnot cytotoxi andc is capable when introduced to cell ofs exhibiting a great subceler lula routir ng efficiency from an extracell ular space to a subcellular compartment of an endoplasm retiic culum and/or cytosol as compared to the subcellular routing efficienc ayof reference molecule, such as, e.g., the twentieth and/or twenty-f cellirst -targeti molengcules.
Further Embodiments of Embodiment Sets #1—#1 1 157. 157. 157. id="p-157" id="p-157" id="p-157" id="p-157" id="p-157" id="p-157" id="p-157" id="p-157" id="p-157" id="p-157"
[157] In certai embodin ment of sEmbodiment Sets #2 to #11, the Shiga toxin effect polypeptor ideis fused to the binding region, either direct orly indirect suchly, as, e.g., via a linker known to the skilled worker. 158. 158. 158. id="p-158" id="p-158" id="p-158" id="p-158" id="p-158" id="p-158" id="p-158" id="p-158" id="p-158" id="p-158"
[158] In certa embodiin ment of sEmbodiment Sets #2 to #11, the cell-target ing molecule comprises a molecula moietr locay ted carboxy-termi to thenal carboxy­ terminus of the Shiga toxin Al fragment region. 159. 159. 159. id="p-159" id="p-159" id="p-159" id="p-159" id="p-159" id="p-159" id="p-159" id="p-159" id="p-159" id="p-159"
[159] In certai embodin ment of sEmbodime ntSets #2 to #11, the Shiga toxin effect polypor epti hasde a Shiga toxin Al fragme derivednt region having a carboxy terminus and further comprises a disrupt edfurin-cleava motige atf the carboxy­ terminus of the Al fragme region.nt -52-WO 2016/196344 PCT/US2016/034778 53 160. 160. 160. id="p-160" id="p-160" id="p-160" id="p-160" id="p-160" id="p-160" id="p-160" id="p-160" id="p-160" id="p-160"
[160] In certain embodimen ofts Embodiment Sets #2 to #11, the cell-target ing molecule of the prese ntinvention, or a polypeptide component thereof, compris aes carboxy-termi endopnal, las micreticulum, retention/retr signalieval motif of a member of the KDEL family. For certa furthein embodimer nts, the carboxy- termina endoplasmicl reticulum retention/ret signarieval motifl is selected from the group consist ingof: KDEL, HDEF, HDEL, RDEF, RDEL, WDEL, YDEL, HEEF, HEEL, KEEL, REEL, KAEL, KCEL, KEEL, KGEL, KHEL, KLEL, KNEL, KQEL, KREL, KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL, KDEF, KKEL, HADE, HAEL, HIEL, HNEL, HTEL, KIEL, HVEL, NDEL, QDEL, REDL, R\ L L.
RTDL, RTEL, SDEL, IDEL, SKEL, STEL, and EDEL. In certai furthen r embodiments, the cell-targetin molegcule of the prese ntinvention is capable when introduced to cells of exhibiti ngcytotoxicity that is great thaner that of a reference molecule, such as, e.g., a twenty-sec cell-ond target moleculeing consisting of the cell-targetin molegcule except for it does not compris anye carboxy-termin al, endoplasmic reticulum retention/ret signarieval motifl of the KDEL family. In certain further embodiments, the cell-target moleculing ofe the present invention is capable of exhibiting a cytotoxicity with bette optimizr ed, cytotoxic potenc such,y, as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or great cytoter oxici as tycompared to a refere ncemolecul suche, as, e.g., the twenty-second cell-targeti molecng ule. In certain furthe embodimer nts, the cytotoxicity of the cell-target moleingcule of the present invention to a population of targe positt ive cells is 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold or great thaner the cytotoxici of thety twenty- second cell-target moleingcule to a second population of targe positt ive cells as assayed by CD50 values. 161. 161. 161. id="p-161" id="p-161" id="p-161" id="p-161" id="p-161" id="p-161" id="p-161" id="p-161" id="p-161" id="p-161"
[161] In certa embodimein nts of Embodiment Sets #2 to # 11, the Shiga toxin effector polypeptide further compris ates leas onet insert ored embedded, heterologous epitope. 162. 162. 162. id="p-162" id="p-162" id="p-162" id="p-162" id="p-162" id="p-162" id="p-162" id="p-162" id="p-162" id="p-162"
[162] In certain embodiments of Embodiment Sets #2 to #11, the Shiga, toxi n effector polypeptide furthe comprisr ates leas one,t two, or three disrupted, endogenous, B-cell and/o CD4+r T-cell epitope regions. In certain further embodiments, the Shiga toxin effecto polypeptider comprises a disrupti onof at least one, two, or three endogenous B-cell, and/or T-cell epitopes and/o epitoper region s.
In certai furthen embodimer nts, the Shiga toxin effecto polypeptider further compris ates lea stone disrupted, endogenous B-cell, and/or CD4+ T-cell epitoperegion which does not overlap wit hat lea stone inserted or embedded, heterologou s epitope. 163. 163. 163. id="p-163" id="p-163" id="p-163" id="p-163" id="p-163" id="p-163" id="p-163" id="p-163" id="p-163" id="p-163"
[163] In certai embodin ment ofs Embodiment Sets #2 to #11, the amino-terminus of the Shiga toxi effectn polypeptior isde at and/or proxim toal an amino-term inusof a polypept idecomponent of the cell-targeti molengcule In. certai furthern embodiments the ,binding region is not located proxim alto the amino-termi ofnus the cell-targeti molngecule relative to the Shiga toxin effector polypeptide. In certai furthen embodimr ents, the binding region and Shiga toxin effector polypeptide are physical arrangedly or oriented within the cell-targeti molngecule such that the binding region is not located proxim toal the amino-termi ofnus the Shiga toxi n effector polypepti de.In certain furthe embodimr ents, the binding region is located within the cell-targeti molngecule more proxima to lthe carboxy-termi ofnus the Shiga toxin effector polypepti thande to the amino -terminus of the Shiga toxi n effector polypeptide For. certai furthern embodiments, the cell-target moleculing e of the present inventio is nnot cytotox andic is capable when introduced to cell ofs exhibiting a greater subcellular routing efficiency from an extracell spaceular to a subcellula comparr tme ofnt an endoplasm retiic culum and/or cytosol as compared to the subcellula routir ng efficiency of a reference molecule, such as, e.g., a twent y- third cell-target molingecule having an amino-termin andus comprising the binding region and the Shig atoxin effector polypept idewhich is not positioned at or proxim toal the amino-term inusof the third cell-targeti molecng ule. For certai n further embodiments, the cell-target molingecule of the present inventio exhibin ts cytotoxici witty hbetter optimized, cytotoxic potency such, as, e.g., 4-fold, 5-fol d,6­ fold, 9-fold, or greater cytotoxi ascit compy ared to the cytotoxici of thety twenty- thir cell-td argeti molengcule. For certa furtin her embodiments the ,cytotoxici of ty the cell-targeti molngecule of the present invention to a population of target positive cell iss 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fol dor greater than the cytotoxici of thety twenty-t hirdcell-targeti molngecule to a second populat ionof target positive cel lsas assayed by CDSO values. In certai furthern embodiments, the twenty-t hirdcell-targeti molengcule does not compri seany carboxy-termi nal, endoplasm reticicul umretention/ret signarieva motill off the KDEL family. 164. 164. 164. id="p-164" id="p-164" id="p-164" id="p-164" id="p-164" id="p-164" id="p-164" id="p-164" id="p-164" id="p-164"
[164] In certai embodimn ents of Embodiment Sets #2 to #11, the Shiga toxin effector polypepti furtde her comprises a disrupti onin the B-cell and/or T-cel l epitope region selected from the group of natively positioned Shiga toxin A Subunit -54-WO 2016/196344 PCT/US2016/034778 55 regio nsconsisting of: 1-15 of SEQ ID NO: 1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26 37 of SEQ ID NO:3; 27-37 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO 2: 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190 ofSEQ IDNO:1 or SEQ IDNO:2; 179-191 ofSEQ ID NO:3; 204 ofSEQ ID NO:3; 205 ofSEQ ID NO:1 or SEQ ID NO:2. and 210-218 ofSEQ ID NO:3; 240-260 ofSEQ ID NO:3; 243-257 ofSEQ ID N():l or SEQ ID NO:2; 254-268 of SEQ ID NO: I or SEQ ID NO:2; 262-278 ofSEQ ID NO:3; 281-297 ofSEQ ID NO:3; 285-293 of SEQ ID NO: 1 or SEQ ID NO:2; 4-33 of SEQ ID NO: 1 or SEQ ID NO:2; 34-78 ofSEQ ID NO:1 or SEQ ID NO:2; 77-103 ofSEQ ID NO:1 or SEQ IDN0:2; 128-168 ofSEQ IDNO:1 or SEQ ID NO:2; 160-183 of SEQ ID NO: 1 or SEQ ID NO:2: 236-258 of SEQ ID NO: 1 or SEQ ID NO:2: and 274-293 of SEQ ID NO: 1 or SEQ ID NO:2; or the equivale regnt ion in a Shiga toxin A Subunit or derivati therve eof. In certain further embodiments, there is no disruption which is a carboxy-termina truncationl of amino acid residues that overlap with part or all of at leas onet disrupted, endogenous B-cel, and/orl T-cell epitope and/or epitope region. [ 165] In certain embodiments of Embodiment Sets #2 to # 11, the Shiga toxin effector polypept idefurther compris aes mutation, relati tove a wild-type Shiga toxin A Subunit in, the B-cell immunogeni aminoc, acid residue selected from the group of natively positioned Shiga toxin A Subunit amino acid residues: L49, D197, D198, R204, and R205. 166. 166. 166. id="p-166" id="p-166" id="p-166" id="p-166" id="p-166" id="p-166" id="p-166" id="p-166" id="p-166" id="p-166"
[166] In certain embodimen ofts Embodiment Sets #2 to #11, the embedde ord inserte heted, rologous T-ce,ll epitope disrupt thes endogenous B-cell, and/or T-cell epitope region is selected from the group of natively positioned Shiga toxin A Subunit regio nsconsist ingof: (1) 1—15 of SEQ ID NO: 1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; and 53-66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; or the equivale regiont inn a Shiga toxin A Subunit or derivative there of,wherein there is no disrupti onwhich is an amino-termi truncanal tion of sequence thats overlap with part or all of at leas onet disrupt edepitope region; (11) 94-115 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3: 141-153 ofSEQ ID NO:1 or SEQ ID NO:2; 140-156 ofSEQ ID NO:3: 179- WO 2016/196344 PCT/US2016/034778 56 190 of SEQ ID NO: 1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2: and 210-218 of SEQ ID NO:3; and (iii) 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO:1 or SEQ ID NO:2; 254- 268 of SEQ ID NO: 1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO: 1 or SEQ ID NO:2; or the equivalent regio n m a Shiga toxin A Subunit or derivative thereof. 167. 167. 167. id="p-167" id="p-167" id="p-167" id="p-167" id="p-167" id="p-167" id="p-167" id="p-167" id="p-167" id="p-167"
[167] In certain embodiments of Embodiment Sets #2 to #11, the Shiga, toxin effector polypeptide compris aes mutation, relat iveto a wild-type Shiga toxin A Subunit, m the B-cell and/or T-cell epitope region selected from the group of natively positioned Shiga toxin A Subunit region conss ist ingof: (i) 1-15 of SEQ ID NO: 1 or SEQ ID NO: 2; 3-14 of SEQ ID NO: 3; 26-3 7 of SEQ ID NO: 3; 27-3 7 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2: 42-48 of SEQ ID NO:3; and 53-66 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; or the equivale regint on in a Shiga toxin A. Subunit or derivative thereof, wherein there is no disruption which is an amino-terminal truncation of sequences that overlap with part or all of at least one disrupt edepitope region; (ii) 94-115 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO: 1 or SEQ ID NO:2; 140- 156 of SEQ TDNO:3; 179-190 of SEQ TDNO:1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2; and 210- 218 of SEQ ID NO:3; and (iii) 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO:1 or SEQ ID NO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO:1 or SEQ ID NO:2; or the equivalen regiot inn a Shiga toxin A Subunit or derivati thereve of,where in ther ise no disruption which is an amino-terminal truncation of sequences that overlap with part or all of at leas onet disrupted epitope region. 168. 168. 168. id="p-168" id="p-168" id="p-168" id="p-168" id="p-168" id="p-168" id="p-168" id="p-168" id="p-168" id="p-168"
[168] In certain embodiments of Embodiment Sets #2 to #11, the Shiga toxin effector polypept idecompris aes disrupti onof at leas onet endogenous epitope regi on select from,ed the group of natively positioned Shiga toxin A Subunits consisting of: 94-115 of SEQ IDNO:1, SEQ IDNO:2, or SEQ IDNO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO: 1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2; or 210-218 of SEQ ID NO:3. 169. 169. 169. id="p-169" id="p-169" id="p-169" id="p-169" id="p-169" id="p-169" id="p-169" id="p-169" id="p-169" id="p-169"
[169] In certain embodiments of Embodiment Sets #2 to # 11, the Shiga toxin effector polypept idedoes not comprise a heterologous MHC, cla ss!-restricte T- d,WO 2016/196344 PCT/US2016/034778 57 cell epitope MHC. clas !-res stricted, T-cell epitopes are known in the art or can be predicted by the skilled worke r.Hie term heterologous refers to MHC cla ss1- restricte T-ced, ll epitopes whic hare not natively prese ntin wild-type Shiga toxin A Subunits such, as, e.g., the wild-type Shiga toxin A Subunit whic his most closely related to the Shiga toxin effector polypeptide of interest. 170. 170. 170. id="p-170" id="p-170" id="p-170" id="p-170" id="p-170" id="p-170" id="p-170" id="p-170" id="p-170" id="p-170"
[170] In certain embodiments of Embodiment Sets #2 to #11, the Shiga toxin effector polypeptide compris disruptionses of at leas four,t five, six, seven, eight or, more endogenous B-cell, and/or T-cell epitope regions. 171. 171. 171. id="p-171" id="p-171" id="p-171" id="p-171" id="p-171" id="p-171" id="p-171" id="p-171" id="p-171" id="p-171"
[171] In certain embodiments of Embodiment Sets #2 to #11, one or more disruptions compris anes amino acid residue substitution relat iveto a wild-type Shiga, toxin A Subunit. 172. 172. 172. id="p-172" id="p-172" id="p-172" id="p-172" id="p-172" id="p-172" id="p-172" id="p-172" id="p-172" id="p-172"
[172] In certain embodiments of Embodiment Sets #2 to #11, one or more endogenous, B-cell and/o T-cer ll epitope regions compris aes plurality of amino aci d residue substituti onsrelat iveto a wild-type Shiga toxin A Subunit. 173. 173. 173. id="p-173" id="p-173" id="p-173" id="p-173" id="p-173" id="p-173" id="p-173" id="p-173" id="p-173" id="p-173"
[173] In certain embodiments of Embodiment Sets #2 to #11, at leas one,t two, thre ore, four disruptions comprise a pluralit of yamino acid residue substitutions in the endogenous B-cell, and/or T-cell epitope regio relatn iveto a wild-type Shiga, toxin A Subunit. 174. 174. 174. id="p-174" id="p-174" id="p-174" id="p-174" id="p-174" id="p-174" id="p-174" id="p-174" id="p-174" id="p-174"
[174] In certain embodiments of Embodiment Sets #2 to #11, at leas onet disruption compris ates leas one,t two, thre four,e, five, six, seven, eight, or more amino acid residue substituti onsrelat iveto a wild-type Shiga toxin A Subunit, and optionally wherei at.n leas onet substitution occurs at the natively positioned Shiga toxin A Subunit amino acid residue selected form the group consisting of: 1 of SEQ ID NO;1 or SEQ ID NO:2: 4 of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3; 6 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3: 8 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3: 11 of SEQ ID NO: I, SEQ ID NO:2, or SEQ ID NO:3; 12 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 33 of SEQ ID NO: 1 or SEQ ID NO 2: 43 of SEQ ID NO: 1 or SEQ ID NO:2; 44 of SEQ ID NO: 1 or SEQ ID NO:2; 45 of SEQ ID NO: 1 or SEQ ID NO:2; 46 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 47 of SEQ ID NO: 1 or SEQ ID NO:2; 48 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3: 49 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 50 of SEQ ID NO: 1 or SEQ ID NO:2: 51 of SEQ ID NO:1 or SEQ ID NO:2; 53 of SEQ ID NO:1 or SEQ ID NO:2: 54 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 55 of SEQ ID NO: I or SEQ WO 2016/196344 PCT/US2016/034778 58 ID NO:2: 56 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 57 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID XO 3: 58 of SEQ ID NO:1, SEQ ID X():2. or SEQ ID NO:3; 59 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 60 of SEQ ID NO: I or SEQ ID NO:2; 61 of SEQ ID NO: I or SEQ ID NO:2; 62 of SEQ ID NO: 1 or SEQ ID NO:2; 84 of SEQ ID NO: 1 or SEQ ID NO:2; 88 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO: 3; 94 of SEQ ID NO: 1, SEQ ID NO :2, or SEQ ID NO:3; 96 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 104 of SEQ ID NO: I or SEQ ID NO:2; 105 of SEQ IDNO:1 or SEQ IDNO:2; 107 of SEQ ID N():L SEQ IDNO:2, or SEQ ID NO:3; 108 of SEQ ID NO: 1 or SEQ ID NO:2: 109 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 110 of SEQ ID NO: 1 or SEQ ID NO: 2; 111 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 112 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 147 of SEQ ID XO: L SEQ ID NO:2, or SEQ ID NO:3; 154 of SEQ ID NO:1 or SEQ ID NO:2; 179 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 180 of SEQ ID NO:1 orSEQIDNO:2; 181 of SEQ IDNO:1 or SEQ IDNO:2; 183 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 184 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 185 ofSEQIDNO:! orSEQIDNO:2; 186 of SEQ IDNO:1, SEQ H)XO:2 or SEQ TDNO:3; 187 of SEQ IDNO:1 or SEQ IDNO:2; 188 of SEQ IDNO:1 or SEQ ID NO:2; 189 of SEQ ID NO:1 or SEQ ID NO:2; 197 of SEQ ID NO:3: 198 of SEQ ID NO: 1 or SEQ ID NO:2; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2; 247 of SEQ ID NO: I or SEQ ID NO:2: 247 of SEQ ID NO:3; 248 of SEQ ID NO: 1 or SEQ ID NO:2; 250 of SEQ ID NO:3: 251 of SEQ ID NO: 1 or SEQ ID NO:2; 264 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID N():3; 265 of SEQ ID NO:1 or SEQ ID NO:2; and 286 of SEQ ID NO:1 or SEQ ID NO:2; or the equivalent amino acid residue in a Shiga toxin A Subunit or derivati therve eof. In certa in furthe embodr iment ats, leas twot disruptions each comprise at least one amino aci d residue substituti onsrelati tove a wild-type Shiga toxin A Subunit selected form the group consisting of: 1 of SEQ ID NO: 1 or SEQ ID NO:2; 4 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 8 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3: 11 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3: 33 of SEQ ID NO: 1 or SEQ ID NO:2; 43 of SEQ ID NO: 1 or SEQ ID NO:2; 45 of SEQ ID NO: 1 or SEQ ID NO:2; 47 of SEQ ID NO: 1 or SEQ ID NO:2; 48 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQ ID NO: 1 or SEQ ID NO:2: 53 of SEQ ID NO: 1 or SEQ ID NO:2: 55 of SEQ ID NO: 1 WO 2016/196344 PCT/US2016/034778 59 or SEQ ID NO:2; 58 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID N():3; 59 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 60 of SEQ ID NO: 1 or SEQ ID NO:2; 61 of SEQ ID NO: 1 or SEQ ID NO:2; 62 of SEQ ID NO: 1 or SEQ ID NO:2: 94 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 96 of SEQ ID NO: 1, SEQ ID NO:2. or SEQ ID NO:3; 109 of SEQ ID NO: L SEQ ID NO:2, or SEQ ID NO:3; 110 of SEQ ID NO: 1 or SEQ ID NO:2; 112 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 147 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 179 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID N():3; 180 of SEQ ID NO:1 or SEQ ID NO:2; 181 of SEQ ID NO: I or SEQ ID NO:2; 183 of SEQ ID NO: 1, SEQ ID SEQ ID NO:2, or SEQ ID NO:3; 184 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 185 of SEQ ID NO: 1 or SEQ ID NO:2; 186 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NOv: 187 of SEQ ID NO: I or SEQ ID NO:2; 188 of SEQ ID NO: 1 or SEQ ID NO:2; 189 of SEQ ID NO: 1 or SEQ ID NO:2: 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2; 247 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:3: 250 of SEQ ID NO:3; 264 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 265 of SEQ ID NO: I or SEQ ID NO:2; and 286 of SEQ ID NO: 1 or SEQ ID NO:2; or the equivale aminont acid residue in a Shiga toxin A Subunit or derivati therve eof. 175. 175. 175. id="p-175" id="p-175" id="p-175" id="p-175" id="p-175" id="p-175" id="p-175" id="p-175" id="p-175" id="p-175"
[175] In certa embodimein nts of Embodiment Sets #2 to #11, the Shiga toxin effector polypeptide compris disrupties onof at leas thret endogenouse, B-ce, ll and/o T-cer ll epitope regions selected from the group of consisting of: (i) 1-15 of SEQ ID NO: I or SEQ ID NO:2; 3-14 of SEQ IDNO:3; 26-37 of SEQ IDNO:3; 27-37 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; and 53-66 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or the equivalent region in a Shiga toxin A Subunit or derivati thereof,ve wherein there is no disruption which is an amino-terminal truncat ofion amino acid residues that overlap with part or all of at leas onet disrupted, endogenou B-ces, ll and/o T-cer ll epitope region; (ii) 94-115 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO: 1 or SEQ ID NO: 2; 179-191 of SEQ ID NO: 3; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2; and 210-218 of SEQ ID NO:3; and (iii) 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO: 1 or SEQ ID NO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO: 1 or SEQ ID NO:2; or the equivalent region in a Shiga toxin A Subunit or derivative there of,wherei theren is noWO 2016/196344 PCT/US2016/034778 60 disruption which is a carboxy-termi truncanal tion of amino acid residues that overlap with part or all of at least one disrupted, endogenous, B-cell and/o T-cer ll epitope and/or epitope region. 176. 176. 176. id="p-176" id="p-176" id="p-176" id="p-176" id="p-176" id="p-176" id="p-176" id="p-176" id="p-176" id="p-176"
[176] In certa embodimein nts of Embodiment Sets #2 to #11, the Shiga toxin effector polypeptide compris dises ruptions of at lea sttwo, endogenous, B-cell and/or T-cell epitope regions, wherein each disruptio comprisn onees or more amino acid residue substitutions, and wherein the endogenou B-ces, ll and/or T-cell epitope regio nsare selected from the group of natively positioned Shiga toxin A Subunit regio nsconsisting of: 3---14 of SEQ II) NO:3; 26-37 of SEQ ID NO:3; Tl-31 of SEQ ID NO: 1 or SEQ ID NO 2: 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; or the equivale regint on in a Shiga toxin A Subunit or derivative thereof. 177. 177. 177. id="p-177" id="p-177" id="p-177" id="p-177" id="p-177" id="p-177" id="p-177" id="p-177" id="p-177" id="p-177"
[177] In certain embodimen ofts Embodiment Sets #2 to #11, the embedded or inserted, heterologous T-ce,ll epitope does not disrupt any endogenous, B-cell and/o r CD4+ T-cell epitope regio desn cribe hered in. 178. 178. 178. id="p-178" id="p-178" id="p-178" id="p-178" id="p-178" id="p-178" id="p-178" id="p-178" id="p-178" id="p-178"
[178] In certain embodiments of Embodiment Sets #2 to #11, at leas onet disruption compris onees or more amino acid residue substituti onsrelat iveto a wild- type Shiga toxin A Subunit is selected from the group consisting of: D to A, D to G, D to V, D to L, D to I, D to F, D to S, D to Q, D to M, D to R, E to A, E to G, E to V, E to L, E to I, E to F, E to S, E to Q, E to N, E to D, E to M, E to R, F to A, F to G, F to V, F to L, F to I, G to A, G to P, H to A, H to G, H to V, H to L, H to I, H to F, H to M, I to A, I to V, I to G, I to C, K to A, K to G, K to V, K to L, K to I, K to M, K to FI, L to A, L to V, L to G, L to C, N to A, N to G, N to V, N to L, N to I, N to F, P to A, P to G, P to F, R to A, R to G, R to V, R to L, R to I, R to F, R to M, R to Q, R to S, R to K, R to H, S to A, S to G, S to V, S to L, S to I, S to F, S to M, T to A, T to G, T to V, T to L, T to I, T to F, T to M, T to S, V to A, V to G, ¥ to A, Y to G, Y to V, Y to L, Y to I, Y to F, Y to M, and Y to T. In certai furthen r embodiments, the one or more amino aci dresidue substitutions relative to a wild- type Shiga toxin A Subunit is selected from the group consisting of: D to A, D to G, D to V, D to L, D to I, D to F, D to S, D to Q, E to A, E to G, E to V, E to L, E to I, E to F, E to S, E to Q, E to N, E to D, E to M, E to R, G to A, H to K. H to G, H to V, H to L, H to I, II to F, H to M, K to A, K to G, K to V, K to L, K to I, K to M, K to FI, L to A, L to G, N to A, N to G, N to V, N to L, N to I, N to F, P to A, P to G, P to F, R to A, R to G, R to V, R to L, R to I, R to F, R to M, R to Q, R to S, R to K, R WO 2016/196344 PCT/US2016/034778 61 to H, S to A, S to G, S to V, S to L, S to I, S to F, S to M, T to A, T to G, T to V, T to L, T to L T to F, T to M, T to S, Y to A, Y to G, Y to V, Y to L, Y to I, Y to F, and Y to M. 179. 179. 179. id="p-179" id="p-179" id="p-179" id="p-179" id="p-179" id="p-179" id="p-179" id="p-179" id="p-179" id="p-179"
[179] In certa embodimein nts of Embodiment Sets #2 to #11, at leas onet of the disruption(s) compris onees or more amino acid residue substitutions relat iveto a wild-type Shiga toxin A Subunit selected from the group consist ingof: KI to A, G, V, L, I, F, M and H; T4 to A, G, V, L, I, F, M, and S; D6 to A, G, V, L, I, F, S, Q and R; S8 to A, G, V, I, L, F, and M; T9 to A, G, V, I, L, F, M, and S; S9 to A, G, V: L, I, F, and M; KI 1 to A, G. V, L, I, F, M and H; T12 to A, G, V, L L, F. M, S. and K: S12 to A, G, V, I, L, F, and M; S33 to A, G, V, L, I, F, M, and C; S43 to A, G, V, L, I, F, and M; G44 to A or L; S45 to A, G, V, L, I, F, and M; T45 to A, G, V, L, I, F, and M; G46 to A and P; D47 to A, G, V, L, I, F, S, M, and Q; N48 to A, G, V, L, M and F; L49 to A, V, C, and G; Y49 to A, G, V, L, I, F, M, and T; F50 to A, G, V, L, I, and T; A51 ; D53 to A, G, V, L, I, F, S, and Q; V54 to A, G, I, and L; R55 to A.
G, V, L, I, F, M, Q, S, K, and H; G56 to A and P; 157 to A, G, V, and M; L57 to A, V, C, G, M, and F; D58 to A, G, V, L, I, R S. and Q; P59 to A, G, and F; E60 to A, G, V, 1 . I, F, S. Q. N, D, M, T, and R; E61 to A, G, V, L, I, F, S. Q, N, D, M, and R; G62 to A; R84 to A, G, V, L, I, F, M, Q, S, K, and H; V88 to A and G; 188 to A, V, C, and G; D94 to A, G, V, L, I, F, S, and Q; S96 to A, G, V, I, L, F, and M: T104 to A, G, V, L, 1, F, M; and N; A105 to L; T107 to A, G, V, L, 1, F, M, and P; S107 to A, G, V, L, I, F, M, and P; LI 08 to A, V, C, and G; S109 to A, G, V, I, L, F, and M; T109 to A, G, V, I, L, F, M, and S; G110 to A; S112 to A, G, V, L, I, F, and M; DI 11 to A, G, V, L, I, F, S, Q, and T; SI 12 to A, G, V, L, I, F, and M; D141 to A, G, V, L, I, F, S, and Q; G147 to A; V154 to X and G. R179 to A, G, V, L, 1, F, M, Q, S, K, and H; T180 to A, G, V, L, I, F, M, and S; T181 to A, G, V, L, I, F, M, and S; D183 to A, G, V, L, I, F, S, and Q; DI84 to A, G, V, L, I, R S, and Q; LI85 to A, G, V and C; SI86 to A, G, V, I, L, F, and M; G187 to A; RI88 to A, G, V, L, 1, F, M, Q. S, K, and H; S189 to A, G, V, I, L, F, and M; D197 to A, G, V, L, I, F, S. and Q; DI98 to A, G, V, L, I, F, S, and Q; R204 to A, G, V, L, I, F, M, Q, S, K, and H; R205 to A, G, V, L, I, R M, Q, S, K and H; S247 to A, G, V, I, L, F, and M; Y247 to A, G, V, L, I, F, and M; R248 to A, G, V, L, 1, F, M, Q, S, K, and H; R250 to A, G, V, L, I, F, M, Q, S, K, and H; R251 to A, G, V, L, I, F, M, Q, S, K, and H; D264 to A, G, V, L, I, F, S, and Q; G264 to A; and T286 to A, G, V, L, I, F, M, and S.[ 180] For certai embodimn ents of Embodiment Sets #2 to #11, the cell-targeti ng molecul ofe the present invention is capable when introduce to da chordat of e exhibiting improved in vivo tolerabilit and/ory stabil itycompared to a reference molecule, such as, e.g., a twenty-fourth cell-target molingecule consisti ofng the cell-target molingecule except for all of its Shiga toxi effen ctor polypept ide component(s) each comprise a wild-type Shiga toxi Aln fragment and/or wild-type Shig atoxin furin-cleav sitage eat the carboxy terminus of its Al fragment region. In certai furthen embodimr ents, the Shiga toxin effector polypept ideis not cytotoxic and the molecula moietr isy cytotoxic. 181. 181. 181. id="p-181" id="p-181" id="p-181" id="p-181" id="p-181" id="p-181" id="p-181" id="p-181" id="p-181" id="p-181"
[181] In certa embodimin ents of Embodiment Sets #2 to #11, the binding region and Shig atoxin effect polypeptor ideare linked togeth either, er direct orly indirectl y. [ 182] In certa embodimin ents of Embodiment Sets #2 to #11, the binding region comprises at leas onet peptide and/or polypeptide. In certai furthern embodiments, the binding region is or compris anes immunoglobulin-t bindingype region In. certai furthen embodimr ents, the binding region compris aes polypept ideselected from the group consisti of:ng an autonomous Vh domain, single-domai antin body fragment (sdAb), nanobody, heavy chain-antibody domain derived from a cameli d (VhH or Vh domain fragment heavy-c), hain antibody domain derived from a cartilaginous fish (VhH or Vh domai fragmen nt), immunoglobul newin antigen receptor (IgNAR), VNAR fragment, single-chain variable fragme (scFv),nt antibody variable fragment (Fv), complement determary ini regionng 3 fragment (CDR3), constrain FR3-CDed R3-FR4 polypept ide(FR3-CDR3-FR4), Fd fragment, smal l modular immunopharmaceuti (SMIP)cal domain, antigen-binding fragme (Fab),nt Armadil repeatlo polypeptide (ArmRP), fibronectin-derived 10th fibronec typetin III domai (10Fn3),n tenascin type III domai (TNfn3),n ankyrin repeat moti domain,f low-density-lipoprotein-receptor-der A-domai (LDLR-Anived ), lipocali (antin cali n), Kunitz domai n,Protein-A-deri Zved domai n,gamma- Bcrystalline-derived domain, ubiquitin-deriv domaied n,Sac7d-deri vedpolypept ide(affitin), Fyn-derived SH2 domain, miniprotei C-typen, lectin-l domainike scaffold engine, ered antibody mimic, and any genetical manipully ated counterp artsof any of the foregoing which retai bindingn functionality. 183. 183. 183. id="p-183" id="p-183" id="p-183" id="p-183" id="p-183" id="p-183" id="p-183" id="p-183" id="p-183" id="p-183"
[183] For certai embodin ment of sEmbodiment Sets #2 to # 11, the cell-targeting molecul ofe the present invention is capable of exhibiting (i) a catalytic activ ityleve l comparabl to ae wild-type Shiga toxin Al fragment or wild-type Shiga toxin effector -62-polypeptid (ii)e, a ribosome inhibition activi withty a half-maxim inhialbitory concentration (IC50) value of 10,000 picomola or rless, and/or (iii) a significant level of Shiga toxin catalytic activity. 184. 184. 184. id="p-184" id="p-184" id="p-184" id="p-184" id="p-184" id="p-184" id="p-184" id="p-184" id="p-184" id="p-184"
[184] For certai embodimn ents of Embodiment Sets #2 to #11, the cell-targeting molecul ofe the present inventio and/orn its Shiga toxin effector polypepti isde capable of exhibiting subcellula routingr efficiency compara toble a reference cell­ target molecing ule comprising a wild-type Shiga toxi Aln fragment or wild-type Shiga toxin effector polypepti and/orde capable of exhibiting a significant level of intracell routularing activi toty the endoplasm retiic culum and/or cytosol from an endosom staal rti locatng ionof a cell. 185. 185. 185. id="p-185" id="p-185" id="p-185" id="p-185" id="p-185" id="p-185" id="p-185" id="p-185" id="p-185" id="p-185"
[185] For certai embodimen nts of Embodiment Sets #2 to #11, where by administration of the cell-targeti molengcule of the present invention to a cell physical coupledly wit hthe extracellu targetlar biomolecule of the cell-targeti ng molecul’s bindinge region, the cell-target molingecule is capable of causing death of the cell In. certai furthern embodiments admi, nistrati of theon cell-targeting molecule of the invention to tw odifferent populatio ofns cell types whic hdiffer wit h respect to the presence or leve ofl the extracell targetular biomolecul thee, cel­ l target moling ecule is capable of causing cell deat toh the cell-types physical ly coupled wit han extracell targetular biomolecule of the cytotoxi cell-c targeting molecul’s bindie ng region at a CD50 at leas threet time ors les thans the CD50 to cell types whic hare not physical couplely witd han extracell targetular biomolecule of ■ the cell-target molingecule’s binding region. For certa embodin iments whereby, administrati of theon cell-targeti molengcule of the present invention to a first population of cell whoses members are physical coupledly to extracell targetular biomolecul ofes the cell-target moleingcule’s binding region, and a second population of cell whoses members are not physical coupledly to any extracel lular target biomolecule of the binding region, the cytotoxic effect of the cell-targeti ng molecule to members of said first population of cell rels ati tove members of said second population of cell iss at lea st3-fold great er.For certai embodimn ents, whereby administrati of theon cell-targeti molengcule of the present inventio to na first populations of cell whoses members are physical coupledly to a significa nt amount of the extracell tarularget biomolec uleof the cell-targeti molecng ule’s binding region and, a second population of cell whoses members are not physical ly coupled to a significant amount of any extracell targetular biomolec uleof the -63-WO 2016/196344 PCT/US2016/034778 64 binding region, the cytotoxic effect of the cell-targetin molecg ule to members of said first population of cells relati tove member ofs said second population of cells is at leas 3-folt gread ter. For certa embodimein nts, where byadministrat ofion the cell- targeting molecule of the present inventi onto a first population of target biomolec ulepositive cell ands, a second population of cells whose member dos not express a significa amountnt of a targ biomoleculet of ethe cell-targetin moleculeg ’s bindin gregio atn a cellular surface, the cytotoxic effect of the cell-targetin g molecule to members of tli efirst population of cells relat iveto members of tli e second population of cells is at leas 3-foldt greater. 186. 186. 186. id="p-186" id="p-186" id="p-186" id="p-186" id="p-186" id="p-186" id="p-186" id="p-186" id="p-186" id="p-186"
[186] For certa embodin imen ofts Embodiment Sets #2 to #11, the cell-targetin g molecule of the prese ntinvention is capable when introduced to cel lsof exhibiting a cytotoxic withity a half-maxima inhibil tory concentrati (CD50)on value of 300 nM or less and/o capar ble of exhibiting a significa levent ofl Shiga toxin cytotoxicity. 187. 187. 187. id="p-187" id="p-187" id="p-187" id="p-187" id="p-187" id="p-187" id="p-187" id="p-187" id="p-187" id="p-187"
[187] For certain embodimen ofts Embodiment Sets #2 to #11, the cell-targetin g molecule of the prese ntinvention is capable of delivering an embedded or inserte d, heterologous CD8+, T-cell epitope to a MHC class I presentati pathon way of a cell for cell-surfa presce entati ofon the epitope bound by a MHC clas I smolecule. 188. 188. 188. id="p-188" id="p-188" id="p-188" id="p-188" id="p-188" id="p-188" id="p-188" id="p-188" id="p-188" id="p-188"
[188] In certa embodin imen ofts Embodiment Sets #2 to #11, the cell-target ing molecule compris aes molecular moiety associated wit hthe carboxy-terminus of the Shiga toxin effector polypepti de.In certa embodimein nts, the molecul moiear ty compris ores consis ofts the binding region In. certain embodiments, the molecular moiety compris ates least one ammo acid and the Shiga toxin effector polypeptide is linked to at leas onet amino acid residue of the molecular rnoiety. In certai furthern embodiments, the molecular moiety and the Shiga toxin effector polypeptide are fused forming a. continuous polypeptide. 189. 189. 189. id="p-189" id="p-189" id="p-189" id="p-189" id="p-189" id="p-189" id="p-189" id="p-189" id="p-189" id="p-189"
[189] In certain embodimen ofts Embodiment Sets #2 to #11, the cell-target ing molecule furthe comprisr aes cytotoxic molecular moiet assocy iated with the carboxy-terminus of the Shiga toxin effector polypeptide. For certain embodiments, the cytotoxic molecular moiety is a cytotoxic agent, such as, e.g., a small molecule chemotherapeu agent,tic anti-neoplastic agent, cytotoxic antibiotic, alkyla tingagent, antimetabo topoisomlite, eras inhibitore and/or, tubulin inhibitor known to tire skill ed worker and/or describe hereid . nFor certa furtherin embodiments, the cytotoxic molecul moietyar is cytotoxic at concentra oftions les thans 10,000, 5,000, 1,000, 00 כ, or 200 pM.WO 2016/196344 PCT/US2016/034778 65 190. 190. 190. id="p-190" id="p-190" id="p-190" id="p-190" id="p-190" id="p-190" id="p-190" id="p-190" id="p-190" id="p-190"
[190] In certain embodimen ofts Embodiment Sets #2 to #11, the bindin regg ion is capable of binding to an extracel targelular biomoleculet selected from the group consisting of: CD20, CD22, CD40, CD74, CD79, CD25, CD30, HER2/neu/ErbB2, EGFR, EpCAM, EphB2, prostate-spe membrcific ane antigen, Cripto, CDCP1, endoghn, fibrobla activatst protein,ed Lewis-Y, CD 19, CD21, CSV SLAMF7, CD33, CD52, CD 133, CEA, gpA33, mucin, TAG-72, tyrosine-prote kinasein transmemb ranereceptor (RORl or NTRKR1), carbonic anhydrase IX, fola bindingte protein, gangliosi GD2,de gangliosi GD3,de ganglioside GM2, ganglioside Lewis - Y2, VEGFR, Alpha Vbeta 3,Alpha5betal, ErbBl/EGFR, Erb3, c-MET, IGF1R, EphA3, TRAIL-RI, TRAIL-R2, RANK, FAP, tenasc CD64,in, mesothel BRCA1,in, MART-l/MelanA, gplOO, tyrosinas TRP-1,e, TRP-2, MAGE-1, MAGE-3, GAGE- 1/2, BAGE, RAGE, NY-ESO-1, CDK-4, beta-cateni MUM-n, 1, caspase-8, KIAA0205, HPVE6, SART-1, FRAME, carcinoembry antigen,onic prostat speecific antige prostan, stetem, cell antigen, huma naspartyl (asparaginyl) beta-hydroxyla se, EphA2, HER3/ErbB-3, MUC1, MART-l/MelanA, gplOO, tyrosinase associa ted antigen, HPV-E7, Epstein-Barr virus antigen, Bcr-Abl, alpha-fetoprote antigein n, 17-Al, bladder tumor antigen, CD38, CD15, CD23, CD45 (prote tyrosin ine phosphatas receeptor type C), CD53, CD88, CD129, CD183, CD191, CD193, CD244, CD294, CD305, C3AR, FceRIa gale, ctin- IL-1R9, (interleu1kin- receptor ), mrp-14, NKG2D ligand, programmed death-ligand 1 (PD-L1), Siglec- 8,Siglec-10 , CD49d, CD13, CD44, CD54, CD63, CD69, CD123, TLR4, FceRIa, IgE, CD107a, CD203c, CD 14, CD68, CD80, CD86, CD105, CD115, F4/80, TLT-3, galecti n-3, CD! la-c GITRL,, MHC class I molecule, MHC clas IIs molecule (optiona lly complexe withd a peptide), CD284 (TLR4), CD107־Mac3, CD195 (CCR5), HLA- DR, CD 16/32, CD282 (TLR2), CD11c, and any immunogenic fragment of any of the foregoing. 191. 191. 191. id="p-191" id="p-191" id="p-191" id="p-191" id="p-191" id="p-191" id="p-191" id="p-191" id="p-191" id="p-191"
[191] In certain embodimen ofts Embodiment Sets #2 to #11, the binding region is linked, either directly or indirectl to y,the Shiga toxin effector polypeptide by at least one covalent bond which is not a disulfide bond. In certain further embodiments, tire bindin regg ion is fused, eithe direcr tly or indirect toly, the carboxy-termi ofnus tire Shiga toxin effector polypeptide to form a single continu, ous polypeptide. In certain further embodiment thes, bindin regg ion is an immunoglobulin-type binding region.WO 2016/196344 PCT/US2016/034778 66 192. 192. 192. id="p-192" id="p-192" id="p-192" id="p-192" id="p-192" id="p-192" id="p-192" id="p-192" id="p-192" id="p-192"
[192] In certain embodimen ofts Embodiment Sets #2 to #11, the disrupted furin- cleava motifge compris onees or more mutations in the minimal fur, m-cleav siteage relat iveto a wild-type Shiga toxin A Subunit. In certain embodiments, the disrupted furin-cleavage motif is not an amino-termina truncationl of sequences that overlap with part or all of at lea stone amino acid residue of the minimal furin-cleavage site.
In certain embodiment thes, mutation in the minima l,furin-cleavage sit eis an ammo aci ddeleti on,insertion, and/or substitution of at leas onet amino acid residue in the R/Y -x -X-R furi ncleava motif.ge In certain furthe embodimer nts, the disrupted furm-cleavage motif compris ates lea stone mutation relat iveto a wild-type Shiga toxin A Subunit, the mutation alter ingat leas onet amino acid residue in the region natively positioned 1) at 248-251 of the A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) or Shiga toxin (SEQ ID NO: 2), or 2) at 247-250 of die A Subunit of Shiga - like toxin 2 (SEQ ID NO:3), or tire equivale aminont acid sequence position in any Shiga toxin A Subunit. In certa furthein embodimer nts, the mutati onis an amino aci dresidue substitut ionof an arginine residue with a non-positively charged, ammo acid residue. 193. 193. 193. id="p-193" id="p-193" id="p-193" id="p-193" id="p-193" id="p-193" id="p-193" id="p-193" id="p-193" id="p-193"
[193] In certain embodimen ofts Embodiment Sets #2 to #11, the cell-target ing molecule of the prese ntinvention is capabl whene introduced to cells of exhibiting cytotoxic compaity rable to a cytotoxicity of a reference molecule, such as, e.g., a twenty-fif cell-th target moleingcule consist ingof the cell-target molecing ule except for all of its Shiga toxin effector polypeptide component( eachs) comprise a wild- type Shiga toxin Al fragment. 194. 194. 194. id="p-194" id="p-194" id="p-194" id="p-194" id="p-194" id="p-194" id="p-194" id="p-194" id="p-194" id="p-194"
[194] In certain embodimen ofts Embodiment Sets #2 to #11, the bindin regiog n compris thees peptide or polypeptide shown in any one of SEQ ID NOs: 83-339. In certain further embodiment thes, bindin gregio comprin ses or consists essentiall of y the polypeptide represente by anyd of the following; amino acid s1-245 of any one of SEQ ID NOs: 33, 64, and 65; 269-513 of SEQ ID NO:40 or SEQ ID NO:80; amino acids 269-520 or 269-521 of any one of SEQ ID NOs: 36, 66, and 67; amino acid s1-232, 1-233, 1-234, 1-235, 1-236, 1-242, 1-243, 1-244, 1-245, 1-246, 1- 252, 1-253, I 254. 1-255, or 1-256 of any one of SEQ ID NOs: 47-119 and 176- 248; ammo acid 269-498s or 269-499 of any one of SEQ ID NOs: 37 39. 68 79. and 81; amino acid 269-49s 9, 269-512, 269-513, or 280-510 of any one of SEQ ID NOs: 34, 35, 41-56, and 82.[195] In certai embodimentsn of Embodimen Sett s#2 to #11, the binding region stericall covery thes carboxy-termi ofnus the Al fragment region. 196. 196. 196. id="p-196" id="p-196" id="p-196" id="p-196" id="p-196" id="p-196" id="p-196" id="p-196" id="p-196" id="p-196"
[196] In certai embodimentsn of Embodiment Set s#2 to #11, the molecul moiear ty sterical coversly the carboxy-term ofinus the Al fragment region. In certai furthern embodiment thes, molecular moiety comprises the binding region. 197. 197. 197. id="p-197" id="p-197" id="p-197" id="p-197" id="p-197" id="p-197" id="p-197" id="p-197" id="p-197" id="p-197"
[197] In certai embodimn ents of Embodiment Sets #2 to #11, the cell-target ing molecule of the present inventi oncompris aes binding region and/or molecular moiet y locate carboxy-d term toinal the carboxy-term ofinus the Shiga toxin Al fragment regio n.In certai furtn her embodiment thes, mas sof the binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater. 198. 198. 198. id="p-198" id="p-198" id="p-198" id="p-198" id="p-198" id="p-198" id="p-198" id="p-198" id="p-198" id="p-198"
[198] In certai embodimentn of sEmbodimen Sett s#2 to #11, the cell-targe ting molecule comprises a binding region wit ha mas sof at leas 4.5t kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater as, long as the cell-target molingecule retains the appropriat levele of the Shiga toxi n biological activi notedty herein (e.g., cytotoxici and/tyor intracell routiularng). 199. 199. 199. id="p-199" id="p-199" id="p-199" id="p-199" id="p-199" id="p-199" id="p-199" id="p-199" id="p-199" id="p-199"
[199] In certain embodiment ofs Embodiment Sets #2 to #11, the binding region is comprised withi an relatively large, molecular moiet compriy sin suchg as, e.g., a molecular moiet wity ha mas sof at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cel­ l target moling ecule retains the appropriate level of the Shiga toxin biologica actl ivit y noted herein. 200. 200. 200. id="p-200" id="p-200" id="p-200" id="p-200" id="p-200" id="p-200" id="p-200" id="p-200" id="p-200" id="p-200"
[200] For certain embodiment of sEmbodiment Sets #2 to #11, the cell-target ing molecule of the present inventi onexhibits low cytotoxic potency (i.e. is not capable when introduce to dcertain positive target cel typesl of exhibiting a cytotoxic greateity r than 1% cel deathl of a cell populati aton a cell-targe moltingecule concentration of 1000 nM, 500nM, 100 nM, 75 nM, or 50 nM) and is capabl whene introduc toed cel lsof exhibiting a greate subcellr ular routing efficiency from an extracel lularspace to a subcellula compartr ment of an endoplasmi retic culum and/or cytosol as compared to the subcellula routingr efficiency of a reference molecule, such as, e.g., a twenty-six celth­ l targeti molng ecule having an amino-terminus and comprisi ngthe binding region and the Shig atoxin effector polypept idewhich is not positioned at or proximal to the amino­ terminus of the thir celd l-target molecing ule. In certain -67-WO 2016/196344 PCT/US2016/034778 68 furthe embodr iment thes, twenty-s ixthcell-targeti molecng ule does not comprise any carboxy-termin endoplasmical, reticulum retention/re trievasigna motifll of the KDEL family. 201. 201. 201. id="p-201" id="p-201" id="p-201" id="p-201" id="p-201" id="p-201" id="p-201" id="p-201" id="p-201" id="p-201"
[201] In certa embodin imen ofts Embodiment Sets #2 to #11, In certain furthe r embodiments, the molecular moiet compriy ses a peptide and/o polypeptr idederived from the Shiga toxin A2 fragment of a natural occuly rr ingShiga toxin. 202. 202. 202. id="p-202" id="p-202" id="p-202" id="p-202" id="p-202" id="p-202" id="p-202" id="p-202" id="p-202" id="p-202"
[202] 'The embodiments of the present invention are not intende tod cover any naturally-occ Shigaurring holotoxin or Shiga toxin A Subunit. In certa in embodiments of Embodiment Sets #2-11, the cell-targeti moleculeng of the prese nt invention does not compris ae natura occurrilly Shigang toxin B Subunit. In certain furthe embodr iment thes, cell-targeti moleculeng of the invention does not comprise any polypept idecomprising or consist ingessentia oflly a functional bindin gdomain of a native Shiga toxin B subunit .Rather, in certa embodimein nts of the cell - targeting molecules of the invention, the Shiga toxin A Subunit derived regions are functionally associa tedwith heterologous bindin regionsg to effectu atecell - targeting. 203. 203. 203. id="p-203" id="p-203" id="p-203" id="p-203" id="p-203" id="p-203" id="p-203" id="p-203" id="p-203" id="p-203"
[203] In certain embodimen ofts Embodiment Sets #2 to #11, the bindin regiog n does not comprise a fragment of huma nCD4 corresponding to amino acid residues 19-183. In certa furthein embodimer nts, the bindin gregion does not comprise a fragment of human CD4, a type-I transmembrane glycoprote Inin. certain further embodiments, the bindin gregio doesn not compris ae fragment of a human immune, cell surfac co-receptor.e 204. 204. 204. id="p-204" id="p-204" id="p-204" id="p-204" id="p-204" id="p-204" id="p-204" id="p-204" id="p-204" id="p-204"
[204] In certain embodimen ofts Embodiment Sets #2 to #11, the cell-target ing molecule of the prese ntinvention does not compris ae carboxy-tenmnal, binding regio comprn isi nga fragment of an immune cell surface receptor. 205. 205. 205. id="p-205" id="p-205" id="p-205" id="p-205" id="p-205" id="p-205" id="p-205" id="p-205" id="p-205" id="p-205"
[205] In certain embodiments of Embodiment Sets #1 to #11, the Shiga toxin effector polypept idecompris ates leas two,t embedded or inserte heterd, ologous epitopes. 206. 206. 206. id="p-206" id="p-206" id="p-206" id="p-206" id="p-206" id="p-206" id="p-206" id="p-206" id="p-206" id="p-206"
[206] In certain embodiments of Embodiment Sets # 1 to #11, the Shiga, toxi n effector polypeptide does not comprise the set of amino acid residue substitutions relat iveto a wild-type Shiga toxin A Subunit selected from the followi setsng :(1) R248H and R251H; (2) R248G and R251G; (3) A246G, S247A, A253G, and S254A; and (4) A246G, S247A, R248G, R251G, A253G, and S254A.WO 2016/196344 PCT/US2016/034778 69 207. 207. 207. id="p-207" id="p-207" id="p-207" id="p-207" id="p-207" id="p-207" id="p-207" id="p-207" id="p-207" id="p-207"
[207] In certain embodiments of Embodiment Sets #1 to #11, the Shiga toxin effector polypeptide does not comprise a deletion of the region natively positioned at 247-252 in a wild-type Shiga toxin A Subunit. In certain embodiments of Embodiment Sets #2-11, the Shiga toxin effector polypeptide does not comprise deletions of the regio nsnatively positioned at 245-247 and 253-255 in a wild-type Shiga toxin A Subunit. 208. 208. 208. id="p-208" id="p-208" id="p-208" id="p-208" id="p-208" id="p-208" id="p-208" id="p-208" id="p-208" id="p-208"
[208] In certain embodiments of Embodiment Sets #1 to #11, the Shiga, toxin effector polypeptide compris onees or more mutations relat iveto a naturally occurring A Subunit of a member of the Shiga toxin famil whichy changes an enzymat activic ityof the Shiga toxin effector polypepti thede, mutation selected from at leas onet amino aci dresidue deletion, insertion, or substituti on.In certai n furthe embodimer nts, the mutation relat iveto the natura occurrilly Ang Subunit reduc esof eliminat a escytotoxic activity of the Shiga toxin effector polypeptide but the Shiga toxin effecto polypeptr ideretains at leas onet othe Shigar toxin effector function, such as, e.g., promoting cellular internaliz atiand/oron directing intracellula routingr to a certain subcellular compartment(s) In certa. furtherin embodiments, the mutation relat iveto the naturally occurr ingA Subunit is selected from at least one amino acid residue substitution, such as, e.g., A231E, R75A, Y77S, YIUS, E167D, R170A, R176K, and/o W203Ar in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. 209. 209. 209. id="p-209" id="p-209" id="p-209" id="p-209" id="p-209" id="p-209" id="p-209" id="p-209" id="p-209" id="p-209"
[209] For certain embodimen ofts Embodiment Sets #1 to #11, the Shiga toxin effector polypeptide is capable of: (i) routing to a subcellular compartment of a cell in which the Shiga toxin effector polypeptide is prese ntselected from the following: cytos ol,endoplasmic reticulum, and lysosome (li); intracellular delivery of the epitope from an early endosoma comparl tment to a proteasome of a cell in which the Shiga toxin effector polypeptide is present; and/or (iii) intracel lulardeliver ofy the epitope to a MHC clas I smolecule from an early endosoma compartml ofent a cel l in which the Shiga toxin effector polypeptide is present. In certai furthern embodiments, the Shiga toxin effector polypeptide is capabl ofe intracellula r delivery of the CD 8+ T-cell epitope for presentat byion a MHC class I molecule on the surfac ofe a cell in which the Shiga toxin effector polypept ideis present. 210. 210. 210. id="p-210" id="p-210" id="p-210" id="p-210" id="p-210" id="p-210" id="p-210" id="p-210" id="p-210" id="p-210"
[210] In certain embodiments, the molecule of the present invention does not comprise, at a position carboxy-termina of the lShiga toxin effecto polypeptr ide and/o ther carboxy-terminus of the Shiga toxin Al fragment region, any additional WO 2016/196344 PCT/US2016/034778 70 exogenou mates rial representi anng antigen and/or heterologous CD8+,, T-cel l epitope-peptide. 211. 211. 211. id="p-211" id="p-211" id="p-211" id="p-211" id="p-211" id="p-211" id="p-211" id="p-211" id="p-211" id="p-211"
[211] In certain embodimen ofts Embodiment Sets #2 to #11, the bindin regiog n does not comprise a ligand. In certain embodiments of Embodiment Sets #2 to #11, the bindin regg ion does not comprise a chemokine or a INF-related apoptos is- inducing ligand (TRAIL) nor a receptor binding fragment thereof. In certain embodiments of Embodiment Sets #2 to #11, the bindin gregion does not comprise a human chemokine or huma nTRAIL nor a receptor binding fragment thereof. In embodiments of Embodiment Sets #2 to #11, the immunoglobulin-type binding region does not comprise a ligand nor a receptor bindin gfragment thereof. In certain embodiments of Embodiment Sets #2 to #11, the immunoglobulin-type binding region does not comprise a chemokine or a TNF-related apoptosis-induc ing ligand (TRAIL) nor a receptor bindin gfragment thereof In. certai embodimen nts of Embodiment Sets #2 to #11, the binding region does not comprise a huma nCC chemokine nor a receptor bindin fragmg ent thereof. In certain embodiments of Embodiment Sets #2 to #11, the bindin regg ion does not comprise the human CC chemokine CCL2 (see Bose S, Cho J et al., rircri Pharm Res 36: 1039-50 (2013)).
In certai embodimen nts of Embodiment Sets #2 to #11, the binding regio doesn not comprise the human, CC chemokine CCL2, nor a recept bindinor gfragment thereof, and a carboxy-term Shigainal, toxin effector polypeptide consist ingof amino acid s 75-247 of StxA. In certain embodiments of the cell-targetin molegcule of the present invention, the binding region does not compris thee human, CC chemoki ne CCL2, nor a receptor binding fragment thereof, fuse dto a carboxy-termi Shiganal, toxin effector polypeptide consisting of ammo acids 75-247 of StxA (SEQ ID NO:2). In embodiments of Embodiment Sets #2 to #11, the binding region does not comprise the huma nTRAIL nor a receptor binding fragment thereof. 212. 212. 212. id="p-212" id="p-212" id="p-212" id="p-212" id="p-212" id="p-212" id="p-212" id="p-212" id="p-212" id="p-212"
[212] Among certain embodiments of the prese ntinvention is a pharmaceut ical composition compri sing any one of the above Shiga toxin effecto polypepr tides of the prese ntinvention and/o anyr one of the above cell-target molecing ules of the present invention; and at leas onet pharmaceutic acceptaally excipieble ntor carrier. 213. 213. 213. id="p-213" id="p-213" id="p-213" id="p-213" id="p-213" id="p-213" id="p-213" id="p-213" id="p-213" id="p-213"
[213] Among certain embodiments of tire present invention is a diagnost ic composition comprising any one of the above cell-targeti molecng ules of the present invention and a detection promoti agent.ng Certa furthein embodimr ents are ceil- targeting molecules of the present invention wherein the detectio promotn ing agent WO 2016/196344 PCT/US2016/034778 71 is a heterologous epitope and the cell-targeti moleculng comprise thees heterologous epitope. 214. 214. 214. id="p-214" id="p-214" id="p-214" id="p-214" id="p-214" id="p-214" id="p-214" id="p-214" id="p-214" id="p-214"
[214] Beyond the Shiga toxin effecto polypeptidesr of the present invention, cell- targeting molecules of the present invention, and compositions thereof, polynucleoti capadesble of encoding a Shiga toxin effector polypeptide or cell- targeting molecule of the present invention are within the scope of the present invent on,i as wel asl expression vectors which, compris ae polynucleot ofide the present invention and host ceils comprisi ngany polynucleot and/oride express ion vector of the prese ntinvention. Host cells comprisin ang express ionvector may be used, e.g., in methods for producing a molecule of the prese ntinvention or a polypeptide componen or tfragment thereof by recombina expressint on. 215. 215. 215. id="p-215" id="p-215" id="p-215" id="p-215" id="p-215" id="p-215" id="p-215" id="p-215" id="p-215" id="p-215"
[215] Among certain embodiments of the prese ntinvention is a method of killing a cell comprisin theg step of contact theing cell with any of the above cell-targetin g mol ecule ofs the present invention or the above pharmaceutica compol sitions of the present invention. In certa embodimein nts, the step of contact theing cell(s occur) s in vitro. In certain other embodiments, the step of contacting the cell( occs) urs or in vivo. In furthe embodimer nts of the cell-kil lingmethods, the method is capable of selective killly ing cell( and/os) celr typesl preferentiall over othery cell( and/os) cellr type whens contacting a mixtur ofe cells which diffe witr hrespect to the extracellular presence and/or express ionleve ofl an extracellular targe biomolt ecul e of the binding region of the cell-target molecing ule. 216. 216. 216. id="p-216" id="p-216" id="p-216" id="p-216" id="p-216" id="p-216" id="p-216" id="p-216" id="p-216" id="p-216"
[216] The present invention further provides methods of treating diseases , disorde and/orrs, conditions in patients comprisin theg step of administeri tong a patient in need there aof therapeutically effecti amountve of a cell-targetin g molecule and/o pharmr aceutica compositionl of the prese ntinvention. In certa in embodiments, the disease, disorder, or condition to be treated using a method of the invention is selected from: a cancer, tumor, growth abnormality, immune disorder, or microbia infecl tion. In certain embodimen ofts thes methods,e the cance tor be treated is select fromed the group consisting of: bone cance breasr, canct er, central/periphera nervousl system cancer, gastrointest cancinaler, germ ceil cancer , glandular cancer, head-neck cancer, hematological cance kidney-urinarr, tracty cancer, liver cance lung/pler, uracance prostatr, cance er, sarcoma, skin cancer, and uterine cancer. In certa embodimein nts of thes methe ods, the immune disorder to be treated is an immune disorder associated wit ha disease selected from the group WO 2016/196344 PCT/US2016/034778 72 consisting of: amyloidosis ankylos, spondyling iti asts, hma, Crohn’s disease, diabetes graft, rejection, graft-versus-ho disease,st Hashimoto’s thyroiditi s, hemolytic uremic syndrome, HIV-related disease lupuss, erythematosus multiple, scleros polyarteriis, nodostis polyarthria, psoriatis, sis psoria, ticarthritis rheuma, toid arthritis scleroderma, septi, shock,c Sjorgr’ens syndrome ulcer, ati colitive ands, vasculitis. 217. 217. 217. id="p-217" id="p-217" id="p-217" id="p-217" id="p-217" id="p-217" id="p-217" id="p-217" id="p-217" id="p-217"
[217] 'The use of any׳ composition of matte ofr the prese ntinvention for the treatme ornt prevention of a cance tumor,r, grow thabnormal and/ority, immune disorder is withi then scop eof the prese ntinvention. Among certa embodimentsin of the prese ntinvention is a cell-target moleingcule of the present invention and/or a pharmaceuti composical tion thereof for the treatment or prevention of a cancer, tumor, growth abnormality, immune disorder and/or, microbial infection. Among certain embodiments of the present invention is the use of a cell-targetin moleculeg of the prese ntinvention and/o pharmaceutr composiical tion thereof in the manufactur of a.e medicament for the treatmen or prevet ntion of a cance tumor,r, growth abnormality, immune disorder, or microbial infection. 218. 218. 218. id="p-218" id="p-218" id="p-218" id="p-218" id="p-218" id="p-218" id="p-218" id="p-218" id="p-218" id="p-218"
[218] Certa embodin imen ofts the cell-target moleculeing ofs the prese ntinvention may be utilized for the delive׳־ ryof additional exogenou mates rial into a cell physicall coupledy wit han extracel targelular biomolecult of ethe cell-targeting molecule of the invention. Additiona thelly, present invention provides a method for delivering exogenou materias to thel inside of a cell(s) comprisin contactig theng cell( s)eit, her in vitro or in vivo, with a cell-targeting molecul phare, maceutic al composition, and/o diagnostr compositionic of the present invention. The present invention furthe provider a smethod for deliveri exogenoung materials to tire inside of a cell(s) in a. patient, the method comprisi theng step of administeri tong the patient a cell-targeti molecng ule of the present invention (wit hor without cytotoxic activity), wherein the targe cetll(s) is physically coupled with an extracel targelular t biomolecul of ethe cell-target molecing ule. 219. 219. 219. id="p-219" id="p-219" id="p-219" id="p-219" id="p-219" id="p-219" id="p-219" id="p-219" id="p-219" id="p-219"
[219] Among certain embodiments of the prese ntinvention is a method of delivering into a cell a T-cell epitope capabl ofe being presented by a MHC clas I s molecule of the cel l,the method comprisin theg step of contacting the cell with the cell-target moleingcule of the present invention which is associa tedwit ha heterologous T-ce,ll epitope and/or a composition thereof (e.g., a pharmaceuti orcal diagnosti composc ition of the present invention).WO 2016/196344 PCT/US2016/034778 73 220. 220. 220. id="p-220" id="p-220" id="p-220" id="p-220" id="p-220" id="p-220" id="p-220" id="p-220" id="p-220" id="p-220"
[220] Among certain embodiments of the present invention is a method for "seeding" a tissue locu withins a chordate the ,method comprising the step of: administerin to theg chordate a cell-targetin molecg ule of the prese ntinvention, a pharmaceuti composical tion of the present invention, and/o ar diagnostic composition of the present invention. In certain further embodiments, the methods of the invention for "seeding" a tissu elocu ares for "seeding" a tissu elocus which compris aes maligna diseasnt, ed, or inflamed tissue. In certain further embodiments, tiie methods of the invention for "seeding" a tissue locus are for "seeding" a tissue locu whichs compris thees tissue selected from the group consist ingof: diseas ed tissue, tumor mass, cancerous growt tumor,h, infecte tisdsue, or abnorm celal lular mass. In certain fiirthe embodimer nts, the methods of the invention for "seeding" a tissue locus comprises administeri tong the chordate the cell-target moleculeing of the invention, the pharmaceutic composial tion of the invention, or the diagnost ic composition of the in vention comprising the heterolog T-ceous,ll epitope select ed from the group consisting of: peptides not natively presented by the target cells of tiie cell-targeti molecng ule in MHC class I complexes pepti, des not natively presen t within any protei expressn byed the targe celt l,peptide nots natively present withi n the proteo meof the targe celt l,peptide nots native presely ntin the extracellu lar microenvironm of entthe site to be seeded, and peptide nots natively present in the tumor mass or infected tissu esite to be targeted. 221. 221. 221. id="p-221" id="p-221" id="p-221" id="p-221" id="p-221" id="p-221" id="p-221" id="p-221" id="p-221" id="p-221"
[221] The use of any composition of matte ofr the prese ntinvention for the diagnosis prognosis,, and/or characteriz ofation a disease, disorder, and/or condition is withi then scope of the present invention. Among certa embodimin ents of the present invention is a metho ofd using a cell-targeti molengcule of tiie present invention comprisin a gdetecti promotion agentng and/o composir tion of the invention (e.g. a diagnos compotic siti on)for the collection of information useful in the diagnosis, prognosis or chara, cteriz ofation a disease, disorder, or condition.
Among certain embodiments of the present invention is the method of detectin a g cell (or subcellular compartment there of)using a. cell-target moleingcule and/o r diagnos compostic ition of the prese ntinvention, the method comprising the steps of contacting a cell wit hthe cell-targeti molengcule and/o diagnosr compositic tion and detecting the presence of said cell-targeti molengcule and/or diagnostic compositi on.
In certai embodn iment thes, step of contacting the cell (s)occurs in vitro. In certain embodiments, the step of contacting the cell(s) occurs in vivo. In certain WO 2016/196344 PCT/US2016/034778 74 embodiments, the step of detectin theg ceil( s)occurs in vitro. In certa in embodiments, the step of detectin theg cell( occs) urs in vivo. In certai furthern embodiments, the m ethod involve thes detection of the location of the cell-targeting molecule in an organism using one or more imaging procedures afte ther administra oftion the cell-targeti moleculng toe said organism. For example, cell- targeting molecules of the invention which incorporat detee ctio promon ting agents as described herein may be used to characteriz diseease ass potentially treata byble a related pharmaceut composical ition of the prese ntinvention. For example, certa in cell-target moleculeing ofs the present invention and compositi onsthereof (e.g. pharmaceuti compocal sitions and diagnosti compc ositions of the present invention ), and methods of the present invention may be used to determine if a patient belongs to a group that responds to a pharmaceuti composical tion of the present inventi on.
For example, certa cellin -targetin moleculeg ofs the prese ntinvention and compositions there mayof be used to identify cells which present a del ivered heterologous epi tope-pept ideon acellular surfa ceand/o tor identify subjects containing ceils which prese nta heterologous epitope-peptide delivered by a cell- targeting molecule of the present invention. 222. 222. 222. id="p-222" id="p-222" id="p-222" id="p-222" id="p-222" id="p-222" id="p-222" id="p-222" id="p-222" id="p-222"
[222] Among certain embodiments of the prese ntinvention is a. method of produci nga molecule of the present invention, the method comprisin theg step of purifying tire molecule of tire invention or a polypeptide componen oft there usingof abacterial cell-wa protell domainin interacti suchon, as, e.g., protei Ln from P. magnus or derivati andves bindin gdomain fragments thereof. In certain furthe r embodiments, the purifying step of the method involves the Shiga toxin effector polypept idecomprisin org consist ingessentiall of anyy one of the polypepti des shown in SEQ ID NOs: 6-32 and 340-383. In certa furtherin embodiments, the purifying step of the method involves the cell-targeti molengcule comprisin org consisting essentially of any one of the polypeptides shown in SEQ ID NOs: 43-82 and 439-513. 223. 223. 223. id="p-223" id="p-223" id="p-223" id="p-223" id="p-223" id="p-223" id="p-223" id="p-223" id="p-223" id="p-223"
[223] Certain embodimen ofts the Shiga toxin effecto polypeptidesr of the present invention may be utilize asd an immunogen or as a component of an immunogen for the immunization and/o vacr cination of a chordate Among. certain embodiments of the prese ntinvention is a. method of immunizing a chordate using a Shiga toxin effector polypeptide of the prese ntinvention, the method comprisin adminisg teri a ng chordate the Shiga toxin effector polypeptide of the invention. In certain further WO 2016/196344 PCT/US2016/034778 75 embodiments, the Shiga toxin effector polypeptide comprises or consis essts entiall y of any one of the polypeptides shown in SEQ ID NOs: 6-32 and 340-383. 224. 224. 224. id="p-224" id="p-224" id="p-224" id="p-224" id="p-224" id="p-224" id="p-224" id="p-224" id="p-224" id="p-224"
[224] Among certain embodiments of the present invention are kits comprisi nga composition of matter of the invention, and optional instrly, uctions for use, additional reagent( and/ors), pharmaceutic deliveral device(s).y The kit may further comprise reagents and oilier tools for detecting a cell type (e.g. a tumor cell) in a sample or in a subject. 225. 225. 225. id="p-225" id="p-225" id="p-225" id="p-225" id="p-225" id="p-225" id="p-225" id="p-225" id="p-225" id="p-225"
[225] These and othe featurr es,aspect ands advantages of the prese ntinvention will become better understo witod hregard to the following description, appended claims, and accompanying figures The. aforementioned elements of the invention may be individual combily ned or removed freely in order to make other embodiments of the invention, without any statement to object to such synthesis or removal hereinafter.
BRIEF DESCRIPTION OF THE FIGURES 226. 226. 226. id="p-226" id="p-226" id="p-226" id="p-226" id="p-226" id="p-226" id="p-226" id="p-226" id="p-226" id="p-226"
[226] Figure 1 depict exemplas 7, ryShiga toxin A Subunit effector polypeptides of the prese ntinvention (numbered 1-5) and cell-targeti molecng ules comprising the same (e.g. ،،2/3" denotes either Shiga toxin effector polypept ide2 or 3). The depictions of exemplar moleculey in sFigure 1 are for illustrat purpoive ses of certai generaln, arrangements of the structura fealture ofs a limite setd of embodiments of the present invention. It is to be understo thatod these exempla7 ry molecules do not intend, nor should any be construed, to be wholly definit iveas to tli earrangement of any structura fealture and/os compor nents of a molecule of the present invention. The relat ivesize ,location, or number of features shown in the schematic of sFigure 1 have been simplifie d.For example, the relat ivepositions of embedded, heterologous epitopes and disruptions of an endogenous, epitope regions are not fixed. Similarly, the total numbers of embedded, heterologous epitopes and disruptions of an endogenous, epitope regions are not fixed. Certai embodimentsn of the molecules of the prese ntinvention comprise a. plurality of disrupted, endogenous, epitope regions in a single Shiga, toxin effector polypepti suchde, as, e.g., disruptions of four, five, six, seven, eight, nine, or more regions wherein; thes e disrupted, endogenous epitope, regions may be distribute througd hout the Shiga toxin effector polypepti includingde, disruptions which overlap with or are within the furin-cleavage motif of the carboxy-termi regnusion of a Shiga toxin Al WO 2016/196344 PCT/US2016/034778 76 fragment derived region (see Table 8, infra). Certai embodimn ents of the present invention comprise disruptions of endogenou epitopes, regions which are carboxy- termina to lthe carboxy-termi ofnus the Shiga toxin Al fragment, or a derivati ve thereof, such as, e.g. at a position carboxy-termina to any ldisrupted furin-cleavage site motif The. schematic in Figurs e1 are not intended to accuratel portry anyay informati regardingon the relat ivesize sof molecular structu resin any embodiment of the prese ntinvention. 227. 227. 227. id="p-227" id="p-227" id="p-227" id="p-227" id="p-227" id="p-227" id="p-227" id="p-227" id="p-227" id="p-227"
[227] Figure 2 graphical showsly the protei syntn hes inhibitionis activitie of s exemplary cell-target molecing ules of the prese ntinvention m vitro and over a range of concentrat Forions. eac hsample molecule, the luminescent intens ityof lucifera se express duringed the assay in relat iveluminesce unitsnt (RLU) was plotted over the logarithm to base 10 of the concentrat of ionthe cell-target moleculeing test edin picomolar. Thes eexempla׳ cell-r} target molecing ules exhibited ribosome inhibition activitie compas rable to a "control" cell-targeting molecul whosee, Shiga toxin effector polypept idecomponen const iste ofd a wild-type Shiga, toxin A Subunit fragment except for it comprise a ddisrupted, furin-cleavage site at the carboxy- termin ofus its Shiga toxin Al fragment region (SLT-1A-FR (SEQ ID NO:5)).
Figur e2 shows that exemplar altery ations to naturally occurring, Shiga toxin A Subunit polypeptide suchs, as, e.g., de-immunizing substitutions and embedded, heterologous T-ce,ll epitopes did, not significantly impair Shiga toxin catalytic activity. 228. 228. 228. id="p-228" id="p-228" id="p-228" id="p-228" id="p-228" id="p-228" id="p-228" id="p-228" id="p-228" id="p-228"
[228] Figures 3-7 and 9-10 graphical showly that exemplar cell-ta}' rgeting molecules of the prese ntinvention SLT-1 A-combo(n)::scFv-(n) exhibite cell-d targe tedcytotoxic compaity rable to "control" cell-targeti moleculeng SLT-1s A- FR::scFv-(n), whose Shiga toxin effector polypeptide component consiste ofd a wild-type Shiga toxin A Subunit fragment excep fort it comprise a ddisrupted, furin- cleava sitege at the carboxy-termi ofnus its Shiga toxin Al fragment region (SLT- 1A-FR (SEQ ID NO:5)). The percent viability of targe positivt cellse for eac hcell type was plotte overd the logarit tohm base 10 of the cell-target moleculeing concentrat adminiion ster to edthe respective ceils. 229. 229. 229. id="p-229" id="p-229" id="p-229" id="p-229" id="p-229" id="p-229" id="p-229" id="p-229" id="p-229" id="p-229"
[229] Figure 3 graphical showsly that the exemplar ceyll-targeti moleculeng SLT- lA-combo7::scFv-l (SEQ ID NO:44) exhibite cytotd oxici to two,ty different cell- types comparable to a "control" cell-target molecing ule SLT-lA-FR:scFv-l (SEQ IDNO:34).WO 2016/196344 PCT/US2016/034778 77 230. 230. 230. id="p-230" id="p-230" id="p-230" id="p-230" id="p-230" id="p-230" id="p-230" id="p-230" id="p-230" id="p-230"
[230] Figure 4 graphical showsly that the exemplar ceyll-targeti molengcule SET- lA-combo7::scFv־l (SEQ ID NO:44) exhibite cytotoxicd toity a target-posit ceivell type comparable to a "■control" cell-target moleculeing SLT-lA-FR::scFv־l (SEQ ID NO:34). The specificit ofy cell-targetin was showng by using the same assay with a cell line negat ivefor cell-surfa expresce sion of a targe biomolecult of escFv-1. In Figur e4 for cell-li H,ne the perce ntviability of targ negativeet cells was plotte overd the logarithm to base 10 of the cell-target moleculeing concentration administe tored tire cells Figure. 4 shows that the cell-targetin molegcule SET-lA-combo7::scFv- 1 (SEQ ID NO:44) did not exhibit cytotoxici to aty targe negativet cell type at the concentra testtionsed. 231. 231. 231. id="p-231" id="p-231" id="p-231" id="p-231" id="p-231" id="p-231" id="p-231" id="p-231" id="p-231" id="p-231"
[231] Figure 5 graphical showsly that the exemplar celly -targetin moleculeg SET-s lA-combol0::scFv-l (SEQ ID NO:47), SLT-lA-combol6;:sc Fv-l(SEQ ID NO:52), and SLT-lA-combol9::sc Fv-l(SEQ ID NO:55) exhibited cytotoxicity to two, di fferent cell-ty compapes rable to a "control" cell-targetin molegcule SLT-1A- FR: :scFv-1 (SEQ ID NO:34). 232. 232. 232. id="p-232" id="p-232" id="p-232" id="p-232" id="p-232" id="p-232" id="p-232" id="p-232" id="p-232" id="p-232"
[232] Figure 6 graphical showsly that the exemplar ceyll-targeti molengcule SET- lA-combol7::scFv-l (SEQ ID NO:53) exhibited cytotoxici to ty'cell-line A. comparable to a "control" cell-targeti molengcule SLT-1A-FR: :scFv-1 (SEQ ID NO:34); wherea SET-lA-s combo 17: :scFv- 1(SEQ ID NO:53) exhibite atted nuate d cytotoxic to itycell-line B as compared to the control. Figur e6 shows that the cell- targeting molecule SET-lA-combo 18::scFv-1 (SEQ ID NO:54) did not exhibit cytotoxici to bothty cell-types test edat concentra uptions to 100 nM. The cytotoxic resuitylts from this assa fory an untarge ted,wild-type Shiga toxi Aln fragment in lieu of a cell-target moleingcule are shown as well. 233. 233. 233. id="p-233" id="p-233" id="p-233" id="p-233" id="p-233" id="p-233" id="p-233" id="p-233" id="p-233" id="p-233"
[233] Figure 7 graphical showsly that the exemplar ceyll-targeti molengcule SET- lA-combo2::scFv (SEQ-2 ID NO:58) exhibite cytotd oxici to celty l-lines B and G comparable to a ־‘control" cell-targeti molengcule SLT-lA-FR::scFv- (SEQ2 ID NO:35); whereas SLT-lA-combo2::scFv-2 (SEQ ID NO:58) exhibited slightly attenuate cytotoxicityd to cell-line A as compared to the contr ol.Figure 7 shows that the cell-targeti molecng ule SLT-lA-combol3::scFv-2 (SEQ IDNO:62) exhibited strongly attenuated cytotoxici to thety three cell-ty testepes asd compared to the control. 234. 234. 234. id="p-234" id="p-234" id="p-234" id="p-234" id="p-234" id="p-234" id="p-234" id="p-234" id="p-234" id="p-234"
[234] Figure 8 graphical showsly that exemplar cell-y target molecing ules of the present invention scFv-3::SLT-lA-combo (SEQ5 ID NO:64) and scFv-3::SLT-lA- WO 2016/196344 PCT/US2016/034778 78 combo6 (SEQ ID NO:65) exhibite celld -target cytotoxicied to tytwo, different , target positive cell types comparable to a ‘־control" cell-targetin moleculeg scFv- 3::SLT-1A-WT (SEQ ID NO:33), whos Shigae toxin A Subunit component was a wild-type Shiga toxin A Subunit fragmen Thet. percent viability of targ posiet tive cel lsfor two, different, cel typesl was plotted over die logarithm to base 10 of the cell-target moleingcule concentrati adminion ster to edthe cells For. cell-line B, both scFv-3 ::SLT-lA-combo5 (SEQ IDNO:64) and scFv-3:: SET-lA-combo6 (SEQ ID NO:65) exhibite cytod toxic potenc asy measured by CD50 values lesser than an equivalent leve ofl the contr ol.The specificity of cell-targeti wasng shown by using the same assa w'ity ha cell line negat ivefor cell-surfa expressce ion of a targe t biomolecul ofe scFv-3. In Figur e8 for cell-li A,ne the perce ntviability of targe t negativ cellse was plotted over the logarit tohm base 10 of the cell-targetin g molecule concentrati admionnistere to tired cells Figur. e8 shows that the cell- targeting molecules scFv-3::SET-lA-combo (SEQ5 ID NO:64) and scFv- 3::SET- lA-combo6 (SEQ ID NO:65) exhibite untargetedd cytotoxicity to a targe negativet cell type comparable to the control. 235. 235. 235. id="p-235" id="p-235" id="p-235" id="p-235" id="p-235" id="p-235" id="p-235" id="p-235" id="p-235" id="p-235"
[235] Figure 9 graphical showsly that the exemplary cell-targetin molecg ules SET- lA-combo7::scFv (SEQ-4 ID NO:66) and SLT-1 A-combo 14: :scFv-4 (SEQ ID NO:67) exhibite cell-tad rgete cytotd oxici to aty targ positet ive ceil type comparable to a "control" cell-targeting molecule SLT-1A-FR: :scFv-4 (SEQ ID NO:36). The specifici ofty cell-targeti wasng shown by using the same assay with a cell line negativ fore cell-surfa expressce ion of a targe biomolecult of escFv-4. In Figure 9 for cell-line E, the percent viability of targe negativt cellse was plotted over the logarithm to base 10 of the cell-target moleculeing concentrat adminision tered to the cells. Figur e9 shows that the cell-targeti moleculeng SET-lAs -combo7: :scFv-4 (SEQ ID NO:66) and SLT-lA-combol4::scFv-4 (SEQ ID NO;67) exhibited untargeted cytotoxicity to a targe negatt ivecell type comparable to the control. 236. 236. 236. id="p-236" id="p-236" id="p-236" id="p-236" id="p-236" id="p-236" id="p-236" id="p-236" id="p-236" id="p-236"
[236] Figure 10 graphical showsly that the exemplar cell-y' target moleculeing SLT-lA-combo8::scFv- (SEQ5 ID NO:69) exhibited cell-targe cytotedtoxi tocit a y targe positt ive cell type comparable to a "control" cell-targeting molecule SLT-1 A- FR::scFv-5 (SEQ ID NO:37). The cell-targeting molecule SLT-lA-combo9::scFv5־ (SEQ ID NO:70) exhibite attend uated cytotoxic to itythis cell line as compared to the control, and SLT-lA-combol I::scFv-5 (SEQ ID NO:71) exhibite veryd low cytotoxic potenc compary toed the control.[237] Figures 11-12 graphical showly the caspase activi inducedty by exemplar y cell-targeti molengcules of the present inventio SLT-1n A-combo(n): :scFv-1 as compared to the "control" cell-targeti molngecule SLT-1 A-FR: :scFv-(n) whose, Shiga toxin effector polypept idecomponent consist ofed a wild-type Shig atoxin A Subunit fragment excep fort it comprise a ddisrupted, furin-cleava sitge eat the carboxy-term ofinus its Shiga toxin Al fragme regionnt (SLT-1 A-FR (SEQ ID NO:5)). The percen caspaset activi wasty plotted over the logarithm to base 10 of the cell-target molingecule concentration administered to the cell s.Figure 11-12s show that the exemplary, cell-target moleingcule SLT-1s A-combo7::scFv-l (SEQ ID NO:44), SLT-1 A-combo 14: :scFv-1 (SEQ ID NO:50), and SLT-1A- combo7::scFv-7 (SEQ ID NO:81) induce casd pase activit comparably to ae control cell-targeti molngecule for at leas onet cel lil ne tested. 238. 238. 238. id="p-238" id="p-238" id="p-238" id="p-238" id="p-238" id="p-238" id="p-238" id="p-238" id="p-238" id="p-238"
[238] Figure 13 shows the relati antigve enici ofties exemplary cell-target ing molecule ofs the present invention and a control cell-targeting molecule by Western blot analysis under denaturi conditng ions using three diff, erent antibodies recognizi Shigang toxin Al fragments Figure. 13 show pictures of multiple replicate gels and membranes. The first lane marked "MW Marker" shows the migration patte ofrn a prote molein cular weight ladd er,and the approxima sizete of eac hladder protei bandn is labeled in kiloDalt (kDa).ons The sampl esloaded and run in lanes numbered 1^4 are indicate in dthe figur legee nd: #1) SLT-1 A::combo7::scFv-l (SEQ ID NO:44); #2) SLT-1 A-FR: :scFv-1 (SEQ ID NO:34); #3 SLT- lA::combol4::sc Fv-l(SEQ ID NO:50); and SLT-lA::combolO::scFv-l (SEQ ID NO:47). The top panel shows pictures of a Coomassie-staine replid cat gel;e the second panel (from the top) show picts ures of replicat meme brane probed with a- SLT-1A pAbl, third panel (from the top) shows pictures of a replic atemembrane probed with a-SLT-1 A pAb2, and the last panel (from the top) shows pictur esof a replicat meme brane probed wit ha-StxA mAbl. Figure 13 shows that the exemplary cell-targeti molengcule SLT-1s A-combo7::scFv-l (SEQ IDNO:44), SLT-1A- combo 10::scFv-1 (SEQ ID NO:47), and SLT-1 A-combo 14: :scFv-1 (SEQ ID NO:50) each have reduced antigenici in tythis assa compy ared to the reference molecule SLT-1 A-FR: :scFv-1 (SEQ ID NO:34). 239. 239. 239. id="p-239" id="p-239" id="p-239" id="p-239" id="p-239" id="p-239" id="p-239" id="p-239" id="p-239" id="p-239"
[239] Figure 14 graphicall showsy the relat iveantigenici ofties exemplary cell­ targeti moleng cules of the prese ntinventio andn a contr cell-tol argeti molengcule by ELISA analysis using two, different antibodie recognizis Shigang toxi Aln -79-WO 2016/196344 PCT/US2016/034778 80 fragments. The normaliz ELISAed absorbanc signale is graphed as a percenta ofge tire control molecule SLT-lA-FR::scFv- (SEQl ID NO:34). Figur e14 shows that the exempla ceryll-targeti moleculeng SLT-lA-s combo7::scFv-l (SEQ ID NO:44), SLT-lA-combolO::sc Fv-l(SEQ ID NO:47), and SLT-lA-combol4::sc Fv-l(SEQ ID NO:50) eac hhave reduced antigenici inty' this assay compared to the control. 240. 240. 240. id="p-240" id="p-240" id="p-240" id="p-240" id="p-240" id="p-240" id="p-240" id="p-240" id="p-240" id="p-240"
[240] Figures 15-16 graphical showly the relat iveimmunogenicities of exemplary cell-target molecing ules of the present invention measured from ser acollect fromed a mammalia modeln and using in-solution ELISA assa ysto detec int the serum of eac hanima thel quantity of antibodies which recognize an administered Shiga toxin A Subunit derived molecule The. reference cell-targeti moleculeng useds for relat ivecomparisons wer eSLT-lA-FR::scFv-(n), whose Shiga toxi effen ctor polypeptide componen consit ste ofd the furin-cleavage resistant, Shiga toxin effect or polypept ideSLT-1A-FR (SEQ ID NO:5). For eac hanima treatmentl group admini stered a cell-target moleingcule of the prese ntinvention, the percentage value ofs the SLT-lA-FR::scFv-(n ref) ere ncemolecule treatment group was calculate by dividingd the avera ELISAge signal for all the subjects in the ceil - targeting molecule treatment group at a given time-point wit hthe average ELISA signal of the subject ins the SLT-1 A-FR::scFv-(n) reference treatmen group.t The percenta ofge the refere ncefor each experimental treatmen groupt was graphed on the Y-axis, and the day of serum collecti wason graphed on the X-axis .The symbols in Figures 15—16 represe thent average signa forl individual subjects in the indicated group, and the error bars indica thete standar errd or of the mea nfor the subjects in tire group at the indicated time-point Figur. es 15-16 show that the exemplary ceil- targeting molecules SLT-lA-combol::scFv (SEQ-l ID NO:43), SLT-1A- combo7::scFv-l (SEQ IDNO:44), SLT-lA-combolO::scFv-l (SEQ IDNO:47), SLT-lA-combol2::sc Fv-l(SEQ IDNO:49), SLT-lA-combol5::scFv-l (SEQ ID NO:51), SLT-lA-combol6::sc Fv-l(SEQ IDNO:52), SLT-lA-combol9::sc Fv-l (SEQ ID NO:55), SLT-lA-combolO::scFv-2 (SEQ ID NO:61), and SLT-IA- combo22::scFv- (SEQ2 ID NO:63) exhibite reducd edimmunogenici inty this assa y relat iveto the appropria refte,ere ncemolecule SLT-lA-FR::scFv-( n)( SEQ ID NO:34or35). 241. 241. 241. id="p-241" id="p-241" id="p-241" id="p-241" id="p-241" id="p-241" id="p-241" id="p-241" id="p-241" id="p-241"
[241] Figure 17 shows the furin-cleava resgeistance of the cell-targe moleculeted SLT-lA-FR::scFv-9, which comprised a Shiga toxin effecto polypeptider comprising a disrupted furin-cleava motif,ge as compared to a near lyidentical,cytotoxic cell, -targ moletedecul comprie sing a wild-type Shiga toxin Al fragment wit ha wild-type furin-cleav siteage (SLT-lA-WT::scFv-9). Figure 17 shows a Coomassie-stai polyacrylned, ami gel afterde electrophor ofesi protes samin ples treat wited heither purified, recombina humannt, furin or variou negats ivecontrol conditions. The lanes of the gel are numbered, and the figure legend indicates pre­ treatment conditions of each cell-target moleedcule sampl priore to loading sample to the gel: the temperat inure degrees Celsius (°C), the pre-treat mentdurati onin hours, and whether any furin was added by denoti ngthe amount of furin activit y units added (U) per microgram (pg) of cell-targ moletedecule (labeled "U/pg furin") or "no furin" for zero U/pg furin added. The first lane marked "MW Marke" rshows the migrati patteon ofrn a prote moleculain weirght ladde andr, the approxima sizete of eac hladder protei bandn is labeled in kiloDaltons (kDa). The figure legend indicates which Shiga toxin effector region was present in each cell-target ed molecule sample per lane either, 1) a wild-type furin site (WT) or 2) a disrupted furin moti (ER).f The treat sampled eswere subject edto 0.5 furin activ ityunits per microgram of cell-targe moltedecul (U/epg furin) at 30 °C for 30 hours. Figure 17 shows SLT-1 A-FR::scFv-9 was resist antto 0.5 furin activi unitty sper microgram of SLT-lA-FR::scFv-9 at 30° C. 242. 242. 242. id="p-242" id="p-242" id="p-242" id="p-242" id="p-242" id="p-242" id="p-242" id="p-242" id="p-242" id="p-242"
[242] Figure 18 graphically shows the specifi bindic ng of an exemplary, cell­ targeti molng ecule of the present invention (SEQ ID NO :82) to target positive cell s as compared to target negati celve ls. The amount of cell-targeti molngecule binding to cell wass calculat as edan integrated, mean fluoresce intensnce ity(iMFI) and graphed versus the concentrati of theon cell-targeti molengcule. Curve fitting of the data was used to produce the line fors the two, target-positi cellve-types tested. 243. 243. 243. id="p-243" id="p-243" id="p-243" id="p-243" id="p-243" id="p-243" id="p-243" id="p-243" id="p-243" id="p-243"
[243] Figure 19 graphica showslly the change in human tumor burdens over time for groups of subjec tsin a murin exenograft model after receiving either an exemplary, cell-targeti molngecul ofe the present invention or a vehicle-only control sample. The tumor burden measured as whole body bioluminescence in photons/second was graphed versus time (days post-tumor implant) Admi. nistrati on of the cell-targeti molngecule SLTl-A-combo7::aCD38-scF (SEQv-l ID NO:82) prevent theed increas ine tumor burden observed for the vehic leonly contr group.ol -81-WO 2016/196344 PCT/US2016/034778 82 DETAILED DESCRIPTION 244. 244. 244. id="p-244" id="p-244" id="p-244" id="p-244" id="p-244" id="p-244" id="p-244" id="p-244" id="p-244" id="p-244"
[244] Ilie present invention is describe mored fully hereinafte usingr illustrative, non-limit ingembodiments, and references to the accompanying figure s.This invention may, however, be embodied in many different forms and should not be construed as to be limite tod the embodiments set forth below'. Rather, thes e embodiments are provid edso that this disclosure is thorough and conveys the scope of the invention to those skilled in the art. 245. 245. 245. id="p-245" id="p-245" id="p-245" id="p-245" id="p-245" id="p-245" id="p-245" id="p-245" id="p-245" id="p-245"
[245] In order that the prese ntinvention may be more readily understood, certa in term ares defined below. Additional definitions may be found withi then detail ed description of the invention. 246. 246. 246. id="p-246" id="p-246" id="p-246" id="p-246" id="p-246" id="p-246" id="p-246" id="p-246" id="p-246" id="p-246"
[246] As used in the specification and the appended claim thes, term "sa,"' "an" and "the" include both singul arand the plural refere ntsunles thes context clearly dictat es otherwise. 247. 247. 247. id="p-247" id="p-247" id="p-247" id="p-247" id="p-247" id="p-247" id="p-247" id="p-247" id="p-247" id="p-247"
[247] As used in the specification and. the append edclaim thes, term, "and/or" when referring to two species, A and B, means at leas onet of A and B. As used in die specification and the appended claims the, term "and/or" when referring to great thaner two species such, as A, B, and C, means at leas onet of A, B, or C, or at leas onet of any combination of A, B, or C (with each species in singula orr multiple possibility). 248. 248. 248. id="p-248" id="p-248" id="p-248" id="p-248" id="p-248" id="p-248" id="p-248" id="p-248" id="p-248" id="p-248"
[248] 'Throughout this specificati theon, wrord "comprise" or variatio suchns as "compris"es or "comprising" will be understood to imply the inclusion of a stated, integ er(or components or group) of intege (orrs components but), not the exclusi on of any other integ er(or components) or group of intege (orrs components). 249. 249. 249. id="p-249" id="p-249" id="p-249" id="p-249" id="p-249" id="p-249" id="p-249" id="p-249" id="p-249" id="p-249"
[249] Throughout this specificati tireon, term "including" is used to mea n "including but not limited, to." "Including" and "including but not limited, to" are used interchangeably. 250. 250. 250. id="p-250" id="p-250" id="p-250" id="p-250" id="p-250" id="p-250" id="p-250" id="p-250" id="p-250" id="p-250"
[250] The term "amino acid residue" or "amino aci"d includes reference to an amino acid that is incorporat intoed, a protein, polypeptide, or peptide The. ter m "polypeptide"' includes any polymer of amino acid ors amino aci dresidues The. ter m"polypeptide sequence" refe rsto a series of amino acid ors amino acid residues which physically comprise a polypeptide. A "protein" is a macromole cule comprising one or more polypepti desor polypeptide "chains." A "peptide" is a. smal polypeptidel of size sles thans about a total of 15 to 20 amino acid residues.
The ter m"amino acid sequenc" refe ers to a serie ofs amino acids or amino acid WO 2016/196344 PCT/US2016/034778 83 residues which physicall comprisey a peptide or polypeptide dependi ngon the length. Unless otherwis indice ate polypeptided, and protei sequencen discloss ed herein are written from left to right representing their order from an amino-termi nus to a carboxy-terminus. 251. 251. 251. id="p-251" id="p-251" id="p-251" id="p-251" id="p-251" id="p-251" id="p-251" id="p-251" id="p-251" id="p-251"
[251] The terms ‘‘amino acid," "amino acid residue," "amino acid sequence," or polypept idesequence include natural occurringly ammo acid (inclus ding L and D isosteriomers) and, unless otherwise limited, also include known analogs of natural amino acids that can function in a simila mannerr as naturally occurri aminong acids such, as selenocysteine pyrrol, ysine, 7V-formylmethionine, gamma- carboxyglutam hydroxyprolinehypusine,ate, pyroglutam acidic and, selenomethionine. The amino acid referres to dherein are describ edby shorthand designations as follows in Table A: TABLE A. Amino Acid Nomenclature Name -letter1-letter Alanine Ala A.
Arginine Arg R Asparagine Asn N Aspartic Acid or Aspartate Asp D Cysteine C Cvs E Glutam Aciic d or GlutamateGlu Gin Glutamine Q Glycine G Gly H Histidine His Isoleucine lie I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan w W Tyrosine Tyr Y Valine Vai V 252. 252. 252. id="p-252" id="p-252" id="p-252" id="p-252" id="p-252" id="p-252" id="p-252" id="p-252" id="p-252" id="p-252"
[252] The phras "econserva substitutiontive " with regard to an amino acid residue of a peptide, peptide region, polypept ideregion, protein, or molecule refers to a change in the amino acid composition of the peptide, peptide region, polypept ide region, protein, or molecule that does not substanti allyalter the functi onand structure of the overall peptide, peptide region, polypeptide region, protein, or WO 2016/196344 PCT/US2016/034778 84 molecule (see Creighton. Proteins: Structures and Molecular Properties QS1. H.
Freeman and Company, New York (2nd ed., 1992))). 253. 253. 253. id="p-253" id="p-253" id="p-253" id="p-253" id="p-253" id="p-253" id="p-253" id="p-253" id="p-253" id="p-253"
[253] For purposes of the prese ntinvention, the phras "ederived from" when referr toing a polypept ideor polypeptide regio mean ns that the polypeptide or polypeptide region compris aminoes acid sequence originas foundlly in a "parenta" l protei andn which may now comprise certain amino acid residue additions, deletions trunc, ation reas,rrangements or other, alterat relations iveto the original polypept ideor polypept ideregio asn long as a certa funcin tion( ands) a structur e(s) of the "parent" moleal cule are substantially conserved. The skilled worke willr be able to identify a parental molecule from whic ha polypeptide or polypept ideregio n was derived using techniques known in the art, e.g., protein sequence alignme nt software. 254. 254. 254. id="p-254" id="p-254" id="p-254" id="p-254" id="p-254" id="p-254" id="p-254" id="p-254" id="p-254" id="p-254"
[254] For purposes of the claimed invention and with rega rdto a Shiga toxin polypept idesequence or Shiga toxin derived polypepti thede, term, "wild-typ" e generally refers to a naturally occurring, Shiga, toxin protei sequencn e(s) found in a living species, such as, e.g., a pathogenic bacterium, wherei thatn Shiga toxin protein sequence( iss) one of the most frequently occurri varing ants 'This. is in contra tost infrequently occurri Shiga,ng toxin protei sequencen that,s while still naturally occurring, are found in less than one percent of individua organisml of sa give nspecies when sampling a statistic powerally ful number of naturally occurri ng individual organis ofms that species which comprise at lea stone Shiga, toxin protein varia nt.A clonal expansion of a natural isola outsidete its natural environme nt (regardles of whethes ther isolate is an organism or molecule comprising biological sequence informati doeson) not alter the natural occlyurr ingrequirem entas long as the clonal expansion does not introduc newe 7 sequence varie notty prese ntin naturally occurri populationsng of that species and/o doesr not change the relati ve proporti onsof sequence variants to each other. 255. 255. 255. id="p-255" id="p-255" id="p-255" id="p-255" id="p-255" id="p-255" id="p-255" id="p-255" id="p-255" id="p-255"
[255] The terms "associated," "associating," "linked," or "linking" with regar tod the claim edinvention refers to the state of two or more compone ntsof a molecule being joined, attached, connected, or otherwise coupled to form a single molecule or the act of makin twog molecule associs ated wit heac hother to form a singl e molecule by creating an associati linkage,on, attachment, and/or any other connection between the two molecules For. example, the term "linked" may refe tor two or more compone ntsassociated by one or more atomi interc acti suchons that a WO 2016/196344 PCT/US2016/034778 85 single molecule is forme andd where thein atom icinteracti mayons be coval ent and/o non-covaler Non-lint. mit ingexamples of coval entassociation betwes en two compone ntsinclude peptide bonds and cysteine-cys teinedisulfid bonds.e Non- limiting examples of non-covalent association betwes en two molecul componear nts include ionic bonds. 256. 256. 256. id="p-256" id="p-256" id="p-256" id="p-256" id="p-256" id="p-256" id="p-256" id="p-256" id="p-256" id="p-256"
[256] For purposes of the prese ntinvention, the term "linked" refer to two or more molecul componear ntsassociate by done or more atomic interacti suconsh, that a single molecule is fanned and wherein the atom icinteracti includesons at leas onet covalent bond. For purposes of the prese ntinvention, the term "linking" refe rsto the act of creating a linked molecule as describe above.d 257. 257. 257. id="p-257" id="p-257" id="p-257" id="p-257" id="p-257" id="p-257" id="p-257" id="p-257" id="p-257" id="p-257"
[257] For purposes of the present invention, the term "fused" refers to two or more proteinaceous components associated by at least one covalent bond which is a peptide bond, regardless of whether the peptide bond involves the participation of a carbon atom of a carboxyl acid group or involves another carbon atom, such as, e.g., the a-carbon, p-carbon, y-carbon, a-carbon, etc. Non-limit ingexamples of tw'0 proteinaceous components fuse dtoget herinclude, e.g., an amino acid pept, ide, or polypept idefused to a polypeptide via a peptide bond such that the resulting molecule is a. singl e,continuous polypeptide For. purposes of the prese ntinvention, the ter m"fusing" refers to the act of creating a fuse dmolecule as described above, such as, e.g., a fusion protein generate fromd the recombina fusinton of geneti c regio nswhich when transl atedproduces a single proteinace molecous ule. 258. 258. 258. id="p-258" id="p-258" id="p-258" id="p-258" id="p-258" id="p-258" id="p-258" id="p-258" id="p-258" id="p-258"
[258] The symbol "::" means the polypeptide regions before and aft erit are physically linked together to form a continuous polypeptide. 259. 259. 259. id="p-259" id="p-259" id="p-259" id="p-259" id="p-259" id="p-259" id="p-259" id="p-259" id="p-259" id="p-259"
[259] As used herein, the terms "expressed," "expressing," or "expresse" ands, grammatica variantl thers eof, refe tor translation of a polynucleoti or nuclede icaci d into a protein. The express proteined rnay remain intracellula becor,me a component of the cell surfa cemembrane or be secrete intod an extracellula space.r 260. 260. 260. id="p-260" id="p-260" id="p-260" id="p-260" id="p-260" id="p-260" id="p-260" id="p-260" id="p-260" id="p-260"
[260] As used herein, cells which express a significant amount of an extracellular targe biomolt ecul at eleas onet cellular surface are "targ positet ive cells" or "targe t+ cells" and are cells physicall coupledy to the specifie extrd, acel lulartarge t biomolecule. 261. 261. 261. id="p-261" id="p-261" id="p-261" id="p-261" id="p-261" id="p-261" id="p-261" id="p-261" id="p-261" id="p-261"
[261] As used herein, the symbol "a" is shorthand for an immunoglobulin- type binding region capable of bindin tog the biomolecul followie theng symbol The. symbol "a" is used to refer to the functional characterist of anic immunoglobuli n-WO 2016/196344 PCT/US2016/034778 86 type bindin gregion based on its ability to bind to the biomolecul followinge the symbol wit ha bindin gaffini descty ribed by a dissociati constanton (KD) of 10-5 or less. 262. 262. 262. id="p-262" id="p-262" id="p-262" id="p-262" id="p-262" id="p-262" id="p-262" id="p-262" id="p-262" id="p-262"
[262] As used herein, the ter m"heavy chain variable (Vh) domain" or "light chai n variable (Vl) domain" respectively refe tor any antibody Vh or Vl domai (e.g.n a human Vh or Vl domain) as wel asl any derivati thereofve retaining at least qualitati antigenve binding ability of the corresponding native antibody (e.g. a humanized Vh or Vl domai deriven fromd a native murine Vh or Vl domain) A. Vh or Vl domain consis ofts a "framework" regio inten rrup byted the thre CDRse or ABRs. The framework regions serve to alig then CDRs or ABRs for specif ic bindin tog an epitope of an antigen. From amino-terminus to carboxy-terminus , both Vh and Vl domain comprises the followi framng ewor (FR)k and CDR regions or ABR region s:FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4; or, similarly , FR1, ABR1, FR2, ABR2, FR3, ABR3, and FR4. As used herei n,the term s "HCDR1," "HCDR2," or "HCDR3" are used to refe tor CDRs 1, 2, or 3, respectivel in ay, Vh domai n,and the term "sLCDR1," "LCDR2," and "LCDR3" are used to refer to CDRs 1, 2, or 3, respectively, in a Vl domai n.As used herein, the term "sHABR1," "HABR2," or "HABR3" are used to refer to ABRs 1,2, or 3, respectivel in ay, Vh domai n,and the terms "LABRI," "LABR2," or "LABR3" are used to refer to CDRs 1, 2, or 3, respectivel m ay, Vl domai n.For camel VidhH fragments, IgNARs of cartilaginous fish VNAR, fragments, certa singlein domain antibodies and, derivatives thereof, there is a single heavy, chain variable domain comprising the same basi carrangem ent:FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. As used herein, the term "sHCDR1," "HCDR2," or "HCDR3" may be used to refer to CDRs 1, 2, or 3, respectivel in ay, single heavy chai varin able domain. 263. 263. 263. id="p-263" id="p-263" id="p-263" id="p-263" id="p-263" id="p-263" id="p-263" id="p-263" id="p-263" id="p-263"
[263] For purposes of the prese ntinvention, the term "effector" means providing a biologica actl ivity such, as cytotoxic biologicality, signali ng,enzymat catalysisic , subcellular routing, and/or intermolec bindingular resulting in an alloste effericct(s ) and/o ther recruitme ofnt one or more factors. 264. 264. 264. id="p-264" id="p-264" id="p-264" id="p-264" id="p-264" id="p-264" id="p-264" id="p-264" id="p-264" id="p-264"
[264] For purposes of the prese ntinvention, the phras es"Shiga toxin effector polypeptide," "Shiga toxin effector polypeptide region," and "Shiga toxin effector region" refe tor a polypeptide or polypeptide region derived from at leas onet Shiga toxin A Subuni tof a member of the Shiga toxin family wherein the polypeptide or polypeptide regio isn capable of exhibiting at lea stone Shiga toxin function. For WO 2016/196344 PCT/US2016/034778 87 example, SEQ IDNOs: 8, 11-27, 29-32, 348, 356-358, 363, 370-371, 373, 378- 438 are derived from StxA and SLT-1A. 265. 265. 265. id="p-265" id="p-265" id="p-265" id="p-265" id="p-265" id="p-265" id="p-265" id="p-265" id="p-265" id="p-265"
[265] For purposes of the prese ntinvention, a Shiga toxin effector function is a biologica actl ivity conferr byed a polypeptide region derived from a Shiga toxin A Subunit. Non-limit ingexamples of Shiga toxin effector functions include promoting cell entry; lipid membrane deformation; promoti cellng ular internalizat ion; stimulating clathrin-mediat endoedcyto diresis; ctin intracg ellula routingr to various intracellula compar rtme suchnts as, e.g., the Golgi, endoplasmic reticulum, and cytos ol;directing intracellu routinglar wit ha cargo; inhibiting a ribosome function(s cata); lyti activic ties, such as, e.g., N-glycosidase activit andy catalytica lly inhibiting ribosome reducings; protein synthes inducingis, caspas actie vity, activat effingector caspase effs, ectuating cytost aticeffects and, cytotoxicity. Shiga toxin catalyti actic vitie include,s for example, ribosome inactivat proteiion, n synthesis inhibition, N-glycosida acsetivity, polynucleotide:adenosine glycosidas e activity, RNAase activity, and DNAase activity. Shiga toxins are ribosome inactivati proteng ins (RIPs). RIPs can depurinate nucleic acids polynucleosi, des, polynucleot rRNA,ides, ssDNA, dsDNA, mRNA (and polyA), and vira nucleicl acid (sees e.g., Barbieri L et al., Biochem J286: 1-4 (1992); Barbie riL et al, Nature 372: 624 (1994); Ling J et al., FEES Lett 345; 143-6 (1994); Barbieri L et al., Biochem J 319: 507-13 (1996); Roncuzzi L, Gasperi-Campa A,ni FEES Lett 392: 16-20 (1996); Stirpe F et al., FEES Lett 382: 309-12 (1996); Barbier Li et al., Nucleic Acids Res 25: 518-22 (1997); Wang P, Turner N, Nucleic Acids Res 27: 1900-5 (1999); Barbier Li et al., Biochim Biophys Acta 1480: 258-66 (2000); Barbie riL et al., J Biochem 128: 883-9 (2000); Brigotti M et al., Toxicon 39: 341-8 (2001); Brigot Mti et al., FASEBJ 16: 365-72 (2002); Bagga S et al., J Biol Chem 278: 4813-20 (2003); Picard D et ah. J Biol Chem 280: 20069-75 (2005)). Some RIPs show antivir actial vity and superoxide dismutase activity (Erice A et al., Antimicrob Agents Chemother 37: 835-8 (1993); Au T et al., FEBSLett 471: 169-72 (2000); Parikh B, Turner N, Mini Rev Med Chem 4: 523-43 (2004); Sharma N et al., Plant Physiol 134: 1712004) 81־)). Shiga toxin catalyti actic vitie haves been observe bothd m vitro and in vivo. Non-limit ingexamples of assa ysfor Shiga toxin effector activity measure various activities, such as, e.g., protein synthesis inhibitory activity, depurination activity, inhibiti onof cell growth, cytotoxici supety,rcoile d DNA relaxa tionactivity, and nuclea activise ty.WO 2016/196344 PCT/US2016/034778 88 266. 266. 266. id="p-266" id="p-266" id="p-266" id="p-266" id="p-266" id="p-266" id="p-266" id="p-266" id="p-266" id="p-266"
[266] As used herein, the retention of Shiga toxin effecto functir onrefers to being capable of exhibiting a leve ofl Shiga toxin functiona activl ity, as measured by an appropriate quantitat assiveay with reproducibil compaity, rable to a wild-type, Shiga toxin effector polypeptide contr (e.g.ol a Shiga toxin Al fragme nt)or cell-targetin g molecule comprisin a gwild-type Shiga toxin effector polypeptide (e.g. a Shiga toxin Al fragme nt)under the same conditions. For the Shiga toxin effector function of ribosome inactivati oron ribosome inhibition, retained Shiga toxin effector function is exhibiting an IC50 of 10,000 pM or less in an in vitro setting, such as, e.g., by using an assa knowny to the skilled worke and/or descrr ibed herein. For the Shiga toxin effector function of cytotoxi incit a targety positive cell-kil assal y,retaine d Shiga, toxin effector function is exhibiting a CD50 of 1,000 nM or less depending, on the cell type and its express ionof the appropriate extracellular targe biomolect ule, as shown, e.g., by using an assa knowny to the skilled worker and/o descr ribe hereid n. 267. 267. 267. id="p-267" id="p-267" id="p-267" id="p-267" id="p-267" id="p-267" id="p-267" id="p-267" id="p-267" id="p-267"
[267] For purposes of the claimed invention, the term "equival"ent with regar tod ribosome inhibition means an empirically measured leve ofl ribosome inhibitor׳■ } activity, as measured by an appropriate quantitat asivesa withy reproducibility, which is reproducibly wuthin 10% or less of the activity of the refere ncemolecule (e.g., the second cel !-targeting molecule or third cell-targeting molecule under) the same conditions. 268. 268. 268. id="p-268" id="p-268" id="p-268" id="p-268" id="p-268" id="p-268" id="p-268" id="p-268" id="p-268" id="p-268"
[268] For purposes of the claimed invention, the ter m"equival"ent wit hregard to cytotoxic meaityns an empirical measly ured leve ofl cytotoxicity', as measure byd an appropriate quantitat assiveay with reproducibil whichity, is reproducibl withiy n % or less of the activity of the reference molecul (e.g.,e the second cell-targeting molecule or third cell-targeti molengcule raider) the same conditions. 269. 269. 269. id="p-269" id="p-269" id="p-269" id="p-269" id="p-269" id="p-269" id="p-269" id="p-269" id="p-269" id="p-269"
[269] As used herein, the ter m"attenuat" withed regard to cytotoxici meansty a molecule exhibits or exhibite a dCD50 between 10-fol tod 100-fold of a CD50 exhibite byd a refere ncemolecule under the same conditions. 270. 270. 270. id="p-270" id="p-270" id="p-270" id="p-270" id="p-270" id="p-270" id="p-270" id="p-270" id="p-270" id="p-270"
[270] Inaccurate IC50 and CD50 value shoulds not be considere whend determining a leve ofl Shiga toxin, effector function activity. For some samples, accura valueste for eithe IC50r or CD50 might be unobtainable due to tire inability to collec thet required data point fors an accura curvete fit. For example, theoretica neitlly,her an IC50 nor CD50 can be determine if greatd thaner 50% ri bosome inhibition or cell death, respectivel doesy, not occur in a concentrat serieion fors a given sample.
Data insufficient to accurately' fit a curve as describe ind the analysis of the datafrom exempla Shigary toxi effen ctor functi onassays, such as, e.g., assays described in the Examples below, should not be considered as represent atiof actve ual Shiga toxin effector function. 271. 271. 271. id="p-271" id="p-271" id="p-271" id="p-271" id="p-271" id="p-271" id="p-271" id="p-271" id="p-271" id="p-271"
[271] A failure to dete ctactivit iny Shiga toxi effecn tor function may be due to improper expressi on,polypepti folding,de and/or protein stabilit rathery than a lac k of cell entr y,subcellular routing, and/or enzymat actiic vity. Assays for Shiga toxi n effect functior ons may not require much polypeptide of the inventi toon measure significant amount ofs Shiga toxin effect functior onactivit Toy. the extent that an underlyi causeng of low or no effect functionor is determined empirical toly relat to e protei expresn sion or stabili ty,one of skil inl the art may be able to compensat fore such factors using prote chemin ist andry molecul engiar neer techniqueing knowns in the art, such that a Shiga toxi functin onal effector activi mayty be restored and measured. As examples impr, oper cell-based expressi mayon be compensa tedfor by using different expressio contron sequences;l and improper polypept idefolding and/or stabili mayty benefi fromt stabilizing terminal sequences or ,compensat ory mutations in non-effec regionstor which stabili theze three-dimensi structonal ureof the molecule. 272. 272. 272. id="p-272" id="p-272" id="p-272" id="p-272" id="p-272" id="p-272" id="p-272" id="p-272" id="p-272" id="p-272"
[272] Certa Shigain toxin effector functions are not easil measurable,y e.g. subcellular routing functions. For example, there is no routine, quantitat assayive to distingui whetsh her the failu reof a Shiga toxin effector polypept ideto be cytotoxic and/or deliver a heterologous epitope is due to improp ersubcellula routr ing, but at a tim ewhen tests are available, then Shig atoxi effen ctor polypeptide mays be analyz fored any significant level of subcellular routing as compared to the appropriat wiled-type Shig atoxin effector polypepti de.However, if a Shiga toxi n effect polypeptor idecomponent of a cell-targeti moleculng ofe the present inventio n exhibits cytotoxi comparablcity or eequival entto a wild-type Shiga toxi An Subunit construct then, the subcellular routing activit levely is inferred to be comparabl or e equivalent, respectively to the, subcellular routing activ ityleve ofl a wild-type Shiga toxi An Subunit construct at leas undert the conditio tesnsted. 273. 273. 273. id="p-273" id="p-273" id="p-273" id="p-273" id="p-273" id="p-273" id="p-273" id="p-273" id="p-273" id="p-273"
[273] When new assay fors individual Shiga toxin functions becom avaie lable , Shiga toxin effector polypeptides and/or cell-targeti molengcules comprising Shiga toxi effen ctor polypeptide mays be analyzed for any leve ofl those Shiga toxin effect functior ons, such as a being within 1000-fold or 100-fold or less the activit y of a wild-type Shiga toxin effector polypept ideor exhibiting 3-fold to 30-fol ord -89-greater activit asy compared to a functiona knockoutl Shig, atoxi effen ctor polypeptide. 274. 274. 274. id="p-274" id="p-274" id="p-274" id="p-274" id="p-274" id="p-274" id="p-274" id="p-274" id="p-274" id="p-274"
[274] Sufficient subcellular routing may be merely deduced by observing a molecul’s e cytotoxic activit levelsy in cytotoxici assaty ys, such as, e.g., cytotoxici assayty baseds on T-cell epitope presentati or onbased on a toxin effector function involving a cytosol ic and/or endoplas miretic culum-loca targetlized, substrate. 275. 275. 275. id="p-275" id="p-275" id="p-275" id="p-275" id="p-275" id="p-275" id="p-275" id="p-275" id="p-275" id="p-275"
[275] As used herei n,the retention of "significant" Shig atoxin effecto functir onrefers to a level of Shiga toxin functional activi ty,as measured by an appropria quantite tat ive assa withy reproducibi comparality toble a wild-type Shig atoxin effector polypepti de contr (e.g.ol a Shiga toxin Al fragment). For in vitro ribosome inhibition, significant Shig atoxi effen ctor functi onis exhibiting an IC50 of 300 pM or less depending on the source of the ribosomes used in the assa (e.g.y a bacteri archaealal, or eukaryoti, (algal,c fungal plant,, or animal) source). This is significantly greate inhibitr ion as compare tod the approxim ateIC50 of 100,000 pM for the catalyti calldisruypted SLT-1A 1-251 double mutant (Y77S/E167D). For cytotoxici in aty target-posi celltive-kill assa iny laborat celory culturel signif, ica Shigant toxi effectorn function is exhibiting a CD50 of 100, 50, 30 nM, or les s,dependi ngon the target biomolecule(s of the) binding region and the cell type, particularly that cel typel ’s expressi and/oron cell-surfa ce representat of theion appropri ateextracel targetlular biomolecule(s) and/or the extracellul epitope(s)ar target byed the molecule being evaluated. This is significant ly great cytoter oxici to thety appropriate, target-posi celltive populati ason compared to a Shig atoxin A Subunit alone (or a wild-type Shig atoxin Al fragment), without a cell targeting binding region, which has a CD50 of 100-10,000 nM, depending on the cell line. 276. 276. 276. id="p-276" id="p-276" id="p-276" id="p-276" id="p-276" id="p-276" id="p-276" id="p-276" id="p-276" id="p-276"
[276] For purpose ofs the prese ntinventi onand wit hregard to the Shiga toxin effect or functi onof a molecule of the present invention the, term "reasonabl active "ity refers to exhibiting at leas at moderate level (e.g. within 11-fold to 1,000-fold) of Shiga toxin effect activitor asy define hereid inn relat ionto a molecule comprising a natural ly occurri Shigng atoxi n,where thein Shiga toxi effecn tor activit is yselecte fromd the group consist ingof: internalizat effiionciency, subcellula routir ng efficiency to the cytosol deli, vered epitope presentat byion a target cell(s) ribos, ome inhibition, and cytotoxici Forty. cytotoxici a reasonablety, level of Shiga toxi effecton actr ivit y includes being within 1,000-fold of a wild-type, Shiga toxin construct such, as, e.g., exhibiting a CD50 of 500 nM or less when a -90-WO 2016/196344 PCT/US2016/034778 91 wild-type Shiga toxin construct exhibits a CD50 of 0.5 nM (e.g. a cell-targeting molecule comprisin a gwild-type Shiga toxin Al fragment). 277. 277. 277. id="p-277" id="p-277" id="p-277" id="p-277" id="p-277" id="p-277" id="p-277" id="p-277" id="p-277" id="p-277"
[277] For purposes of the prese ntinvention and with rega rdto the cytotoxicity of a molecule of the prese ntinvention, the ter m"optimal" refers to a level of Shiga toxin catalytic domai mediatedn cytotoxic thatity is within 2, 3, 4, 5, 6, 7, 8, 9, or 10 -fold of the cytotoxici7 ofty a molecule comprisin wild-typeg Shiga toxin Al fragment (e.g. a Shiga, toxin A Subunit or certa truncain tedvariant theres of)and/or a naturally occurring Shiga toxin. 278. 278. 278. id="p-278" id="p-278" id="p-278" id="p-278" id="p-278" id="p-278" id="p-278" id="p-278" id="p-278" id="p-278"
[278] It should be noted that even if the cytotoxici of aty Shiga toxin effector polypeptide is reduced relat iveto a wild-type Shiga toxin A Subunit or fragment thereof, in practi ce,applications using attenuated, Shiga toxin effector polypepti des may be equall ory' more effective than using wild-type Shiga toxin effector polypepti desbecause the highest potency variant mights exhibit undesirabl effectse which are minimized or reduced in reduced cytotoxic-potency variants Wild-type.
Shiga toxins are very' potent, being able to kill an intoxica celted aftel onlyr one toxin molecule has reached the cytos ofol the intoxicat ceiled or perhaps afte onlyr forty toxin molecule haves been internalized into the intoxicat celled Shiga. toxin effector polypeptides with even considera reducedbly Shiga toxin effector functions, such as, e.g.. subceliula routingr or cytotoxici as ty,compared to wild-type Shiga toxin effector polypepti desmay still be potent enoug forh practical applications, such as, e.g., applications involving targete cell-kid lling, heterologous epitope delivery7, and/o detectionr of specif iccells and thei subcelr iular compartments. In addition, certa reduced-ain cti Shigavity toxin effector polypeptides may' be particularly useful for delivering cargos (e.g. an additional exogenou mates rial or T- cell epitope) to certain intracellular locati onsor subceliula compar rtme ofnts target cells. 279. 279. 279. id="p-279" id="p-279" id="p-279" id="p-279" id="p-279" id="p-279" id="p-279" id="p-279" id="p-279" id="p-279"
[279] The term "selective cytotoxicity" with regard to the cytotoxic activi7 tyof a mol ecule refers to the relati levelve of cytotoxici betwety en a biomol ecule targe t positiv cele populationl (e.g. a targe tedcell-type) and anon-targete bystanded cellr population (e.g. a biomolecul targee negativt cell-te ype), which can be express ased a rat ioof the half-maximal cytotoxic concentrati (CD50)on for a targe tedcell type over the CD50 for an untargeted cell type to provide a metri ofc cytotoxic selectivity7 or indication of the preferentiali of killingty of a targete cedll versus an untarge ted cell.WO 2016/196344 PCT/US2016/034778 92 280. 280. 280. id="p-280" id="p-280" id="p-280" id="p-280" id="p-280" id="p-280" id="p-280" id="p-280" id="p-280" id="p-280"
[280] The cell surfac represente ati and/oron density of a given extracellular target biomolecul (ore extracellular epitope of a given targe biomoleculet may) influence the applications for which certai cell-n target moleculesing of the prese ntinvention may be most suitably used. Differences in cel surfl ace representa and/ortion density of a given target biomolecul betwee en cells may alter, both quantitative and ly qualitatively, the efficienc ofy cellular internaliz atiand/oron cytotoxic potencity ofy a given cell-targetin molegcule of the present invention. The cell surface representati and/oon densr ity of a given targe biomolect ulecan vary greatly among target biomolecul positivee cells or even on tire same cell at differ entpoint ins the cell cycle or cell differentiation. The total cell surface representati of aon given targe biomolecult and/oe ofr certa extrain cellular epitopes of a given targe t biomolecul one a particu celllar or population of ceils may be determined using methods known to the skilled worker, such as methods involv ingfluorescen ce- activated cell sorti ng(FACS) flow7 c> iome 5y: 281. 281. 281. id="p-281" id="p-281" id="p-281" id="p-281" id="p-281" id="p-281" id="p-281" id="p-281" id="p-281" id="p-281"
[281] As used herein, the term "sdisrupted," "disruption," or "disrupting," and grammatica varil ants thereof, with rega rdto a polypept ideregion or feature within a polypeptide refe rsto an altera tionof at leas onet amino acid within the region or composing the disrupte featd ure. Amino acid alterat includeions various mutations, such as, e.g., a deletio inversion,n, insertion, or substituti whichon alter the amino aci dsequence of the polypeptide. Amino acid alterations also include chemica l changes such, as, e.g., the altera tionone or more atoms in an amino aci dfunctional group or the addition of one or more atoms to an amino acid functional group. 282. 282. 282. id="p-282" id="p-282" id="p-282" id="p-282" id="p-282" id="p-282" id="p-282" id="p-282" id="p-282" id="p-282"
[282] As used herein, "de-immunize" dmean reduceds antigeni and/orc immunogeni potentiac afterl administrat to iona chordate as compared to a reference molecule, such as, e.g., a wild-type peptide region, polypept ideregion, or polypeptide. This includes a reduct ionin overall antigenic and/o immunogenir c potenti desalpite the introduc tionof one or more, de novo, antige nicand/or immunogenic epitopes as compared to a refere ncemolecule. For certa in embodiments, "de-immunized" means a molecule exhibite reducedd antigeni city and/o immr unogenici afterty administration to a mamma asl compared to a "parent" moleculeal from which it was derive suchd, as, e.g., a wild-type Shiga toxin Al fragment. In certa embodin iment thes, de-immunized, Shiga toxin effector polypeptide of the present invention is capable of exhibiting a relati antigenicitve v compared to a refere ncemolecule which is reduced by 10%, 20%, 30%, 40%, 50%.WO 2016/196344 PCT/US2016/034778 93 60%, 70%, 80%, 90%, or great thaner the antigenicity of the reference molecule under the same conditions measured by the same assa y,such as, e.g., an assa y known to the skilled worker and/o descr ribed herein like a quantitat ELISAive or Wester blotn analysis. In certain embodiments, the de-immunized, Shiga toxin effector polypeptide of the prese ntinvention is capable of exhibiting a relati ve immunogenicit compay red to a reference molecule which is reduced by 10%, 20%, %, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or great thaner the immunogenicit of they refere ncemolecule under the same conditions measured by the same assay, such as, e.g., an assa knowny to the skilled worker and/o descr ribe d herein like a quantitat meaivesurement of anti-molecul antibodiee produceds in a mammal( afters) receiving parente adminiral stration of the molecule at a given time- point. 283. 283. 283. id="p-283" id="p-283" id="p-283" id="p-283" id="p-283" id="p-283" id="p-283" id="p-283" id="p-283" id="p-283"
[283] The relat iveimmunogenicitie of exempls ary cell-targeti molecng ules were determined using an assay for in vivo antibody response to sthe cell-targeting molecules after repeat parenter, administrationsal over periods of many. 284. 284. 284. id="p-284" id="p-284" id="p-284" id="p-284" id="p-284" id="p-284" id="p-284" id="p-284" id="p-284" id="p-284"
[284] For purposes of the prese ntinvention, the phras "B-cee ll and/o CD4+r T-cell de-immunized" means that the molecule has a reduce antid genic and/o r immunogeni potentic afteal adminisr trati to aon mamma regl ard ingeither B-cell antigenicit or immunoy genicit and/oy CD4+r T-cell antigenicit or immunogenicy ity.
For certain embodiment ־‘B-cells, de-immunized" means a molecule exhibited reduc edB-cell antigeni cityand/or immunogenicit aftery administration to a mammal as compared to a "parenta" molel cule from which it was derived, such as, e.g., a wild-type Shiga toxin Al fragment. For certa embodin iment "CD4+s, T-cell de- immunized" means a molecule exhibited reduced CD4 T-cell antigenicit and/y or immunogenicit aftery administration to a mamma asl compared to a. "parenta" l molecule from whic hit was derived, such as, e.g., a wild-type Shiga toxin Al fragment. 285. 285. 285. id="p-285" id="p-285" id="p-285" id="p-285" id="p-285" id="p-285" id="p-285" id="p-285" id="p-285" id="p-285"
[285] The term, "endogenous" with regard to a B-cell epitope, CD4+ T-cell epitope B-ce, ll epitope region, or CD4+ T-cell epitope regio inn a Shiga, toxin effector polypeptide refers to an epitope present in a wild-type Shiga toxin A Subunit. 286. 286. 286. id="p-286" id="p-286" id="p-286" id="p-286" id="p-286" id="p-286" id="p-286" id="p-286" id="p-286" id="p-286"
[286] For purposes of the prese ntinvention, the phras "CD8+e T-cell hyper- immunized" means that the molecul whene, present inside a nucleat chored, date cell withi an living chordate has ,an increased antigenic and/o immunogenir potentiac lWO 2016/196344 PCT/US2016/034778 94 regarding CD8+ T-ceil antigenicit or immunogenicy ity.Commonly CD8+. T-cell immunized molecules are capable of cellu larinternaliz atito anon early endosoma l compartment of a nucleated, chorda cetell due either to an inherent feature(s or )as a componen oft a cell-targetin molecg ule. 287. 287. 287. id="p-287" id="p-287" id="p-287" id="p-287" id="p-287" id="p-287" id="p-287" id="p-287" id="p-287" id="p-287"
[287] For purposes of the prese ntinvention, the term "heterologous" mean ofs a different source than an A Subunit of a naturally occurr ingShiga toxin, e.g. a heterologous polypeptide is not natural foundly as part of any A Subunit of a native Shiga toxin. The ter m"heterologous" with regard to T-ceil epitope or T-cell epitope-pept componentide of a polypeptide of the prese ntinvention refers to an epitope or peptide sequence which did not initiall occury in the polypeptide to be modified, but which has been added to the polypepti whetherde, added via. the processes of embedding, fusion, insertion, and/or amino acid substitut ionas describe herein,d or by any oilier engineer meaing ns. The resul ist a modified polypeptide comprising a T-cell epitope foreign to the origina unmodifiedl, polypepti i.e,de, the T-cell epitope was not prese ntin the original polypeptide. 288. 288. 288. id="p-288" id="p-288" id="p-288" id="p-288" id="p-288" id="p-288" id="p-288" id="p-288" id="p-288" id="p-288"
[288] The term "embedde" dand grammati variantcal theres withof regard to a T- cell epitope or T-cell epitope-pept compide onen oft a polypeptide of the present invention refers to the internal replacem ofent one or more amino acid withins a polypeptide region wit hdifferent amino acid ins order to generate a new polypept ide sequence sharing the same total numbe rof amino acid residues wit hthe starting polypept ideregion. Thus, the term "embedde" ddoes not include any external, termina fusionl of any additional amino acid peptide,, or polypeptide componen to t tiie starti polypeptng idenor any additional intern inseral tion of any additiona aminol acid residues but, rather includes only substitutions for existing amino acids The. intern replacal ement may be accomplis hedmerely by amino acid residue substitution or by a serie ofs substitutions, deletions, insertions, and/or inversions. If an inserti ofon one or more amino acid iss used, then the equivalent number of proximal amino acids must be delete nextd to the insert ionto result in an embedded T-ceil epitope This. is in contra tost use of the term "inserted"' with regar tod a T- cell epitope contained within a polypeptide of the prese ntinvention to refer to the inserti ofon one or more ammo acid inters nally withi an polypept ideresulting m a new polypeptide having an increas numbered of amino acids residues compared to the starti polypepting de.WO 2016/196344 PCT/US2016/034778 95 289. 289. 289. id="p-289" id="p-289" id="p-289" id="p-289" id="p-289" id="p-289" id="p-289" id="p-289" id="p-289" id="p-289"
[289] The term "inserted" and grammatical variants thereof wit hrega rdto a T-cell epitope contained within a polypeptide of the present invention refe rsto the insertion of one or more amino acid withins a polypeptide result ingin a new7 polypeptide sequence having an increas numbered of amino acid resids ues compar ed to the starting polypeptide. The "pure" insertion of a T-cell epitope-pepti isde when the result ingpolypeptide increas ined length by the number of amino acid residues equivalent to the number of amino acid residues in the entire, insert T-ceed ll epitope-pepti de.Hie phras es"partially inserted," "embedded and inserte" andd, grammatica variantsl thereof with rega rdto a T-cell epitope contained ׳withi an polypeptide of the present invention, refers to when the resulting polypeptide increased in length, but by less than the number of amino acid residues equivalent to the length of die entire, insert T-ceed ll epitope-pepti de.Insertions whe, ther "pure" or "partial," include any of the previous descly ribe insed rtio evenns if othe regior ns of the pol ypeptide not proxi mal to the insertion site within the polypept ideare deleted thereby result ingin a decrea inse the to tal length of the final polypeptide because the fina polypeptidel s till compris anes interna insel rtion of one or more amino acids of a T-cell epitope-pept withinide a polypeptide region. 290. 290. 290. id="p-290" id="p-290" id="p-290" id="p-290" id="p-290" id="p-290" id="p-290" id="p-290" id="p-290" id="p-290"
[290] As used herein, the ter m"T-cel epitopel deliver"ing when describing a functional activity of a molecule means that a molecule provide thes biologic al activity of localizing within a cell to a subcellula compartr ment that is competent to resul int the proteaso clmai eavage of a proteinac eouspart of the molecule v, h ich compris aes T-cell epitope-peptide The. "T-cell epitope deliver"ing function of a molecule can be assayed by observing the MHC presentat ofion a T-cell epitope- peptide cargo of the molecule on a cell surfa ceof a cell exogenously administere d the molecule or in whic hthe assa wasy begun with the cell containing the molecule in one or more of its endosoma compal rtme nts.Generally, the ability of a molecule to delive a rT-cell epitope to a proteaso canme be determined where the initial location of the "T-cell epitope deli vering" molecule is an early endosoma l compartment of a cel l,and then, the molecule is empirical shownly to deliv theer epitope-pept toide the proteasome of the cell How. ever. a "T-cell epitope deliver"ing ability may also be determined where the molecule starts at an extracellular location and is empirical showly m, either directly or indirectly, to deliv erthe epitope into a cell and to proteasome of thes ceil .For example, certain ־ T-cell epitope deliver ing־molecules pas sthrough an endosoma comparl tment of WO 2016/196344 PCT/US2016/034778 96 tiie cel l,such as, e.g. after endocytotic entry into that cel i.Alternative "T-cely,ll epitope delivering" activity may be observe ford a molecule starti atng an extracellular location whereby the molecule does not ente anyr endosoma l compartment of a cell—instead the "T-cell epitope deliver"ing molecule enter a scell and delive ars T-cell epitope-pepti tode proteasomes of the cel l,presumably becaus e the "T-cell epitope deliver"ing molecule directe itsd own routing to a subcelhilar compartment competen to tresul int proteasomal cleavage of its T-cell epitope - peptide component. 291. 291. 291. id="p-291" id="p-291" id="p-291" id="p-291" id="p-291" id="p-291" id="p-291" id="p-291" id="p-291" id="p-291"
[291] For purposes of the prese ntinvention, tire phras "eproximal to an amino- terminus" with refere nceto the position of a Shiga toxin effector polypept ideregio n of a cell-targeti moleculeng of the prese ntinvention refers to a distance wherein at lea stone amino acid residue of the Shiga toxin effector polypeptide region is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more, e.g., up to 18-20 amino acid residues of, an amino-tenninus of the ce l-tarl geting mol ecule as long as the cell-targetin g molecule is capable of exhibiting the appropriate leve ofl Shiga toxin effector functional activit notey hereid (e.g.,n a certain leve ofl cytotoxic potency). Thus for certain embodiments of the prese ntinvention, any amino aci dresidue(s) fuse d amino-termin to theal Shiga toxin effecto polypeptider should not reduce any Shiga toxin effector function (e.g., by sterically hindering a structur e(s)near the amino- termin ofus tire Shiga toxin effecto polypeptr ideregion) such that a function al activity of the Shiga toxin effector polypeptide is reduced below the appropriate activity level require hereid n. 292. 292. 292. id="p-292" id="p-292" id="p-292" id="p-292" id="p-292" id="p-292" id="p-292" id="p-292" id="p-292" id="p-292"
[292] For purposes of the prese ntinvention, the phras "emore proximal to an amino-tenninu" wits hrefere nceto the position of a Shiga toxin effector polypeptide regio withinn a cell-target moleingcule of the present invention as compared to another component (e.g., a cell-targe bindinting, gregion, molecul moietar y,and/or additional exogenou mates rial refers) to a position wherein at leas onet amino acid residue of the amino-termi ofnus the Shiga toxin effecto polypeptider is clos toer the amino-tenninus of a linear, polypeptide component of the cell-target molecing ule of tiie present invention as compared to the othe refr erence component.d 293. 293. 293. id="p-293" id="p-293" id="p-293" id="p-293" id="p-293" id="p-293" id="p-293" id="p-293" id="p-293" id="p-293"
[293] For purposes of the prese ntinvention, the phras "eactive enzymat domaiic n derived from one A Subunit of a member of the Shiga toxin family" refe rsto having the ability to inhibit protein svnthesis via a caiah tic ribosome inactivation mechanism. Ine enzymati actic viti ofes natural occurringly Shiga toxins may be WO 2016/196344 PCT/US2016/034778 97 defined by the ability to inhibit protein translation using assa ysknown to the skill ed worker, such as, e.g., m vitro assa ysinvolving RNA translation in the absenc ofe living cells or in vivo assays involving RNA translation in a living cel l.Using assa ysknow n to the skilled worker and/or described herein, the potency of a Shiga, toxin enzymat actic ivity may be assessed direct byly observing N-glycosidase activity toward ribosomal RNA (rRNA), such as, e.g., a ribosome nicking assay, and/or indirect byly observin inhibitig onof ribosome function and/o proteir n synthesis. 294. 294. 294. id="p-294" id="p-294" id="p-294" id="p-294" id="p-294" id="p-294" id="p-294" id="p-294" id="p-294" id="p-294"
[294] For purposes of the prese ntinvention, tire term "Shiga toxin Al fragment region" refe rsto a polypeptide regio consistn ingessentia oflly a Shiga toxin Al fragment and/or derived from a Shiga, toxin Al fragment of a Shiga toxin. 295. 295. 295. id="p-295" id="p-295" id="p-295" id="p-295" id="p-295" id="p-295" id="p-295" id="p-295" id="p-295" id="p-295"
[295] For purposes of the prese ntinvention, the terms "terminus," "amino- terminus" or, "carboxy-terminus" with rega rdto a cell-targeti molecng ule refe rs generally to the las aminot acid residue of a polypeptide chain of the cell-targetin g molecule (e.g., a single continu, ous polypeptide chain). A cell-targetin moleculeg may compris moree than one polypeptides or proteins and,, thus a, cell-targeting m olecule of the prese ntinvention m ay compri se multiple amino-terminals and carboxy-terminals For example,. the "amino-terminus" of a cell-target moleingcule may be defined by the first amino acid residue of a polypeptide chai repren sentin g the amino-term endinal of the polypepti whichde, is generally characteriz by eda starting, amino acid residue which does not have a peptide bond with any amino aci d residue involv ingthe prima7ry amino group of the starti aminong acid residue or involving the equivale nitrogent forn starti aming no acid residues whic hare members of the clas ofs N -alkylat alphaed amino acid residues Similar. ly,the "carboxy-terminus" of a cell-targetin molegcule may be defined by the last amino acid residue of a polypeptide chain representing the carboxyl-ter endminal of the polypepti whichde, is general charactly eriz by aed fina l,amino acid residue which does not have any amino acid residue linked by a peptide bond to the alpha-car bon of its primary carboxyl group. 296. 296. 296. id="p-296" id="p-296" id="p-296" id="p-296" id="p-296" id="p-296" id="p-296" id="p-296" id="p-296" id="p-296"
[296] For purposes of the prese ntinvention, the term "sterminus," "amino- terminus" or, "carboxy-terminus" with rega rdto a polypeptide region refers to the regional boundaries of that region, regardl ofess whethe additionalr amino acid residues are linked by' peptide bonds outside of that region. In othe wordsr the, terminals of the polypeptide region regardl ofess whether that region is fuse dto WO 2016/196344 PCT/US2016/034778 98 oilier peptides or polypeptides For. example, a fusion protein comprisi twong proteinaceous regions, e.g., a bindin gregio comprisingn a peptide or polypept ide and a Shiga toxin effector polypeptide may, have a Shiga toxin effecto polypeptr ide region with a. carboxy-termi endingnus at amino aci dresidue 251 of the Shiga, toxin effector polypeptide regio desn pite a peptide bond involving residue 251 to an amino acid residue at position 252 representing tire beginning of another proteinaceous region, e.g., the bindin gregion. In this example, the carboxy- terminus of the Shiga toxin effecto polypeptr ideregion refers to residue 251, which is not a terminus of the fusion protein but rather represents an intern regionalal, boundary'. Thus, for polypeptide regions, the terms "terminus," "amino-termi"nus, and "carboxy-term" areinus used to refer to the boundaries of polypeptide regions, whether the boundar isy' a physically terminus or an interna positl, ion embedde d withi an larger polypeptide chain. 297. 297. 297. id="p-297" id="p-297" id="p-297" id="p-297" id="p-297" id="p-297" id="p-297" id="p-297" id="p-297" id="p-297"
[297] For purposes of the prese ntinvention, the phras "ecarboxy-termi regionus n of a. Shiga toxin Al fragment" refe rsto a polypeptide regio derivedn from a naturally occurri Shigang toxin Al fragment, the regio begin nning with a hydrophobic residue (e.g., V236 of StxA-Al and SLT-1AL and V235 of SLT-2A1) that is followed by a hydrophobi rescidue and the regio endingn with the furin- cleava sitege conserve amongd Shiga toxin Al fragment polypeptides and ending at the junction between the Al fragment and tire A2 fragment in native, Shiga toxin A Subunits .For purpos esof the present invention, the carboxy-termina regiol ofn a Shiga toxin Al fragment includes apeptidic region derived from the carboxy- terminus of a Shiga toxin Al fragment polypepti de,such as, e.g., a peptid icregio n comprising or consisting essentiall of they carboxy-terminus of a Shiga toxin Al fragment. Non-limitin examplesg of peptidic regions derived from the carboxy- termin ofus a Shiga toxin Al fragment include the amino acid residue sequences natively positioned from position 236 to position 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, or 251 in StxlA (SEQ ID NO:2) or SLT-1A (SEQ ID NO: 1); and from position 235 to position 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250 in SLT-2A (SEQ ID NO:3). 298. 298. 298. id="p-298" id="p-298" id="p-298" id="p-298" id="p-298" id="p-298" id="p-298" id="p-298" id="p-298" id="p-298"
[298] For purposes of the prese ntinvention, tire phras "eproximal to the carboxy- terminus of an A l fragment polypeptide" with regard to a linked molecul moietar y and/o bindinr regg ion refers to being within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 WO 2016/196344 PCT/US2016/034778 99 amino acid residues from the amino acid residue defining the las rest idue of the Shiga toxin Al fragment polypeptide. 299. 299. 299. id="p-299" id="p-299" id="p-299" id="p-299" id="p-299" id="p-299" id="p-299" id="p-299" id="p-299" id="p-299"
[299] For purposes of the prese ntinvention, the phras "esterically cove rsthe carboxy-tenninus of the Al fragment-derive regiond" includes any molecular moiety of a size of 4.5 kDa or great (e.g.,er an immunoglobulin-type bindin gregion) linked and/or fuse dto an ammo aci dresidue in the carboxy-terminus of the Al fragment-de rivedregion such, as, e.g., the amino acid residue derived from the amino acid residue natively positioned at any one of position 236s to 251 in Stxl A (SEQ ID NO:2) or SLT-1A (SEQ ID NO:1) or from 235 to 250 in SLT-2A (SEQ ID NO:3). For purposes of the prese ntinvention, the phras "esterically cover thes carboxy-termi ofnus the Al fragment-derive regidon" also includes any molecul ar moiety of a size of 4.5 kDa or great (e.g.,er an immunoglobulin-type bindin gregion) linked and/or fuse dto an ammo aci dresidue in the carboxy-terminus of the Al fragment-de rivedregion such, as, e.g., the amino acid residue carboxy-termi to nal the las aminot acid A1 fragment-de riveregiod and/on ther Shiga toxin effect or polypeptide. For purposes of the prese ntinvention, the phras "estericall coversy the carboxy-termi ofnus the Al fragment-derive regidon" also includes any molecul ar moiety of a size of 4.5 kDa. or great (e.g.,er an immunoglobulin-t bindinype gregion) physicall prevey nting cellular recognition of the carboxy-tenninu of thes Al fragment-derive region,d such as, e.g. recognit byion the ERAD machine ofry a eukaryotic cell. 300. 300. 300. id="p-300" id="p-300" id="p-300" id="p-300" id="p-300" id="p-300" id="p-300" id="p-300" id="p-300" id="p-300"
[300] For purposes of the prese ntinvention, a bindin gregion, such as, e.g., an immunoglobulin- bindintype gregion, that compris aes polypeptide comprisin atg leas fortt aminoy acids and that is linked (e.g., fused to) the carboxy-terminus of the Shiga toxin effector polypeptide regio comprn isin ang Al fragment-derive regiod n is a molecular moiety which is "stericall coveringy the carboxy-termi ofnus the Al fragment-derive region.d ’’ 301. 301. 301. id="p-301" id="p-301" id="p-301" id="p-301" id="p-301" id="p-301" id="p-301" id="p-301" id="p-301" id="p-301"
[301] For purposes of the prese ntinvention, a bindin gregion such, as, e.g., an immunoglobulin-type bindin gregion, that compris a.es polypeptide comprisin atg leas fortt aminoy acids and that is linked (e.g., fused to) the carboxy-tenninus of the Shiga toxin effector polypeptide region comprisin ang Al fragment-derive regiod n is a molecular moiety "encumbering the carboxy-termi ofnus the Al fragme nt- derived region."WO 2016/196344 PCT/US2016/034778 100 302. 302. 302. id="p-302" id="p-302" id="p-302" id="p-302" id="p-302" id="p-302" id="p-302" id="p-302" id="p-302" id="p-302"
[302] For purposes of the prese ntinvention, the term "Al fragment of a member of lire Shiga toxin famil" yrefer tos the remaining amino-terminal fragment of a Shiga toxin A Subunit after proteol ysisby furi nat the furin-cleavage site conserv amonged Shiga toxin A Subunit sand positione betweend the Al fragment and the A2 fragment in wild-type Shiga toxin A Subunits. 303. 303. 303. id="p-303" id="p-303" id="p-303" id="p-303" id="p-303" id="p-303" id="p-303" id="p-303" id="p-303" id="p-303"
[303] For purposes of the claimed invention, the phras "efurin-cleavage motif at the carboxy-terminus of the A1 fragment region" refers to a specifi c,furin-cleavage motif conserv amonged Shiga toxin A Subunits and bridging the junction between tiie Al fragment and the A2 fragment in natural occurly ring, Shiga toxin A Subunits. 304. 304. 304. id="p-304" id="p-304" id="p-304" id="p-304" id="p-304" id="p-304" id="p-304" id="p-304" id="p-304" id="p-304"
[304] For purposes of the prese ntinvention, the phras "efurin-cleavage site proximal to the carboxy-termi ofnus the Al fragment region" refers to any identifiabl fure,in-cleavage site having an amino acid residue within a distance of less than 1, 2, 3, 4, 5, 6, 7, or more amino acid residues of the ammo acid residue defining the las aminot aci dresidue in the Al fragment region or Al fragment derived region, including a furin-cleava motifge located carboxy-ter minaof an All fragment regio orn Al fragment derived region, such as, e.g., at aposition proximal to the linkage of the Al fragment-de rivedregio ton another component of the molecule, such as, e.g., a. molecul moiear ty of a cell-targeti molengcule of the present invention. 305. 305. 305. id="p-305" id="p-305" id="p-305" id="p-305" id="p-305" id="p-305" id="p-305" id="p-305" id="p-305" id="p-305"
[305] For purposes of the prese ntinvention, the phras "edisrupt edfurin-cleavage motif" refe rsto (i) a specif icfurin-cleava motifge as describe hereid inn Section I-B and (ii) which comprises a mutation and/or truncation that can conf era molecule wit ha reduct ionin furin-cleavage as compared to a refere ncemolecule, such as, e.g., a reduct ionin furin-cleavage reproducibly observed to be 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or less (including 100% for no cleavage) than the furin-cleava of age reference molecule observe ind the same assay under the same conditions. Hie percentage of furin-cleavage as compared to a referenc molee cule can be expressed as a rat ioof cleaved:unclea materiaved ofl the molecule of intere dividedst by the cleavedamclea materialved of the reference molecule )see Examples, supra). Non-limit ingexamples of suitable reference molecule incls ude certain molecule comprs isin a gwild-type Shiga toxin furin- cleava motifge and/or furin-cleava sitege as describe hereind in Section I-B, Section IV-B, and/or the Examples and/o) moleculer useds as refere ncemolecule in sthe Examples below.WO 2016/196344 PCT/US2016/034778 101 306. 306. 306. id="p-306" id="p-306" id="p-306" id="p-306" id="p-306" id="p-306" id="p-306" id="p-306" id="p-306" id="p-306"
[306] For purposes of the prese ntinvention, the phras "efurin-cleavage resist"ant means a molecule or specif icpolypept ideregion thereof exhibits reproducibly less furin cleava thange (i) the carboxy-tenninus of a Shiga toxin Al fragment in a wild- type Shiga toxin A Subunit or (ii) the carboxy-tenninu of thes Shiga toxin Al fragment derived region of construct wherein the natural occlyurr ingfurin-cleavage site nativel positiy oned at the junction between the Al and A2 fragments is not disrupted; as assayed by any availa blemeans to the skilled worker, including by using a method described herein. 307. 307. 307. id="p-307" id="p-307" id="p-307" id="p-307" id="p-307" id="p-307" id="p-307" id="p-307" id="p-307" id="p-307"
[307] For purposes of the prese ntinvention, the phras "eactive enzymat domaiic n derived form an A Subunit of a member of the Shiga toxin family" refers to a polypept idestructure having the ability to inhibit protei synthen sisvia catalytic inactiva tionof a ribosome based on a Shiga toxin enzymat actiic vity. The ability of a molecul strucar ture to exhibit inhibitor acty ivity of protei synthesisn and/or catalytic inactivati ofon a ribosome may be observed using various assa ysknown to the skille worker,d such as, e.g., in vitro assa ysinvolving RNA translati assayson in tiie absence of living cells or in vivo assa ysinvolving the ribosom esof living cells.
For example, using assa ysknown to the skill edworke ther, enzymat actic ivity' of a molecule base don a Shiga toxin enzymat actic ivity'׳ may be assess eddirectly by observing N-glycosidase activity toward ribosom RNAal (rRNA), such as, e.g., a ribosome nicking assa y,and/or indirectl by yobserving inhibition of ribosome function, RNA transla tion,and/o proteinr synthesis. 308. 308. 308. id="p-308" id="p-308" id="p-308" id="p-308" id="p-308" id="p-308" id="p-308" id="p-308" id="p-308" id="p-308"
[308] As used herei withn respect to a Shiga toxin, effector polypepti ade, "combination" describes a Shiga toxin effector polypeptide comprisin twog or more sub-regions wherein each sub-region compris ates leas onet of the following: (1) a disruption in an endogenous epitope or epitope region; (2) an embedded, heterologous T-ce,ll epitope-peptide; (3) an inserted hete, rologous T-ce,ll epitope- peptide; and (4) a disrupted furin-cleava motifge at the carboxy-terminus of an Al fragment region.
Introduction 309. 309. 309. id="p-309" id="p-309" id="p-309" id="p-309" id="p-309" id="p-309" id="p-309" id="p-309" id="p-309" id="p-309"
[309] lire present invention provides various combi, natio Shigan, toxin effector polypepti desand cell-targeting molecules comprisin theg same. Certain embodiments of the Shiga toxin effector polypepti desof the prese ntinvention combine structura elelment ress ulting in two or more properti ines a singl molee cule ,WO 2016/196344 PCT/US2016/034778 102 such as, e.g., the ability to 1) exhibi reducedt antigeni cityand/or immunogenicit as y compared to molecular varia ntslacking that particular combinat ionof elements, 2) exhibit reduced, protease-cleava as comparedge to molecul variaar nts lacking that particula combinatr ionof elements, 3) exhibit reduced non-specif toxicitic toy a multicellula organismr at certain dosages as compar toed molecular variant lacs king that particular combinat ionof elements, 4) deliv eran embedde ord inserted T-cell epitope to the MHC clas Is system a. cell for cell-surface presentation, and/or 5) exhibit potent cytotoxicity. The Shiga toxin effector polypepti desof the present invention may serv ase scaffolds to creat variouse cell-targeting molecule suchs, as, e.g., cell-targete cytotoxic,d, therapeuti molecc ules ce;ll-targete nontoxic,d, delivery' vehicle ands; cell-targete diagnosd, molectic ules.
I. The Gener alStructures of the Shiga Toxin Effector Polypeptides of the Invention 310. 310. 310. id="p-310" id="p-310" id="p-310" id="p-310" id="p-310" id="p-310" id="p-310" id="p-310" id="p-310" id="p-310"
[310] The Shiga toxin effector polypepti d es and cell-targeti m oleculng ofes the present invention comprise at leas one,t Shiga toxin effector polypeptide derived from wild-type Shiga toxin A Subunits but comprise one or more structura l modificati ons,such as, e.g., a mutation like a truncation and/o aminor acid residue substitution( Fors). certa embodin iment thes, prese ntinvention involves the engineerin of gimproved, Shiga toxin A Subunit effecto polypeptidesr comprising the combinat ionof two or more of the followi Shigang toxin effecto polypeptr ide sub-regions: (1) a de-immuniz edsub-region, (2) a protease-cleava resistange sub-t regio nearn the carboxy-termi ofnus a Shiga toxin Al fragment region, and (3) a. T- cell epitope-pept embeide dded or insert sub-region.ed 311. 311. 311. id="p-311" id="p-311" id="p-311" id="p-311" id="p-311" id="p-311" id="p-311" id="p-311" id="p-311" id="p-311"
[311] X Shiga toxin effector polypept ideis a polypeptide derived from a Shiga toxin A Subunit member of the Shiga toxin family that is capabl ofe exhibiting one or more Shiga toxin functions (see e.g., Cheung M et al., Mol Cancer 9: 28 (2010); WO 2014/164693; WO 2015/113005; WO 2015/113007; WO 2015/138452; WO 2015/191764). Shiga toxin functions include, e.g., increas ingcellular internaliza directingtion, subcellular routing from an endosoma compartl ment to the cytosol, avoiding intracellul degradar ation, catalytica inacllytiva tingribosomes and, effectuating cytost aticand/or cytotoxic effects. 312. 312. 312. id="p-312" id="p-312" id="p-312" id="p-312" id="p-312" id="p-312" id="p-312" id="p-312" id="p-312" id="p-312"
[312] The Shiga toxin family of protei toxinsn is composed of various naturally occurring toxins which are structural andly functionally relate e.g.,d, Shiga toxin, Shiga-like toxin 1, and Shiga-like toxin 2 (Johannes L, Romer W, Nat Rev Microbiol WO 2016/196344 PCT/US2016/034778 103 8; 105-16 (2010)). Holotox membersin of the Shiga toxi familn containy targeting domains that preferentia bindlly a specif icglycosphingolip president on the surfa ce of some host cells and an enzymatic domain capabl ofe permanen inactly tivating ribosomes once inside a cell (Johannes L, Romer W, Nat Rev Microbiol 8: 105-16 (2010)). Member ofs the Shiga toxin family shar thee same overall structure and mechanism of action (Engedal N et al., Microbial Biotech 4: 32-46 (2011)). For example, Stx, SLT-1 and SLT-2 display indistinguish enzymatiable actc ivit iny cel l free systems (Head S et al., J Biol Chern 266: 3617-21 (1991); Tesh V et al., Infect Immun 61: 3392-402 (1993); Brigotti M et al., Toxicon 35:1431-1437 (1997)). 313. 313. 313. id="p-313" id="p-313" id="p-313" id="p-313" id="p-313" id="p-313" id="p-313" id="p-313" id="p-313" id="p-313"
[313] The Shiga toxin family encompas setrues Shiga toxin (Stx.) isolated from ־؟. dysenteriae serotype 1, Shiga-like toxin 1 variants (SLT1 or Stxl or SLT-1 or Slt-I ) isolated from serotype of senterohemorr E.hagic coif and Shiga-like toxin 2 varia nts (SLT2 or Stx2 or SLT-2) isolated from serotypes of enterohemorr E.hagic coll.
SLT1 differs by only one amino acid residue from. Stx, and both have been referred to as Verocytotoxins or Verotoxins (VTs) (O’Brie nA, Curr Top Microbiol Immunol 180: 65-94 (1992)). Although SLT1 and SLT2 variants are only about 53-60% similar to eac hother at the primary amino acid sequence level they, shar e mechanisms of enzymati activityc and cytotoxic commonity to the member ofs the Shiga toxin family (Johannes L, Romer W, Nat Rev Microbiol 8: 105-16 (2010)).
Over 39 differ entShiga toxins have been described, such as the defined subtypes Stxla, Stxl c,Stxld, and Stx2a-g (Scheutz F et al., J Clin Microbiol 50: 2951-63 (2012)). Members of the Shiga toxin family are not natural restrily cted to any bacter ialspecies because Shiga-toxin-encod genesing can spread among bacteri al species via horizont geneal trans fer(Strauch E et al., Infect Immun 69: 7588-95 (2001); Bielaszews Mka et al., Appl Environ Micrbiol 73: 3144-50 (2007); Zhaxybay evaO, Doolittle W, Curr Biol 21; R242-6 (2011)). As an exampl ofe interspecies transfer a Shiga, toxin was discovere in ad strai ofn A. haemolyticus isolat fromed a patient (Grotiuz G et al., J Clin Microbiol 44: 3838-41 (2006)).
Once a Shiga, toxin encoding polynucleot enteide rsa. new subspecies or species, the Shiga toxin amino acid sequence is presumed to be capable of developing slight sequence variations due to genetic drift and/o selectiver pressur whilee still maintaining a mechanis ofm cytotoxici commty on to members of the Shiga toxin famil (seey Scheut Fz et al., J Clin Microbiol 50; 2951-63 (2012)).WO 2016/196344 PCT/US2016/034778 104 A. De-immunized, Shiga Toxin A Subuni tEffector Polypeptides 314. 314. 314. id="p-314" id="p-314" id="p-314" id="p-314" id="p-314" id="p-314" id="p-314" id="p-314" id="p-314" id="p-314"
[314] In certa emboin diments, the Shiga toxin effector polypeptide of the present invention is de-immunize suchd, as, e.g., as compared to a wild-type Shiga toxin, wild-type Shiga toxin polypepti and/orde, Shiga toxin effector polypeptide comprising only wild-type polypeptide sequences The. de-immunize Shigad, toxin effector polypeptides of the present invention each compri sea disruption of at least one, putative, endogenous, epitope region in order to reduce the antige nicand/or immunogeni potentic ofal the Shiga toxin effector polypept ideaft eradministra tion of tire polypeptide to a chordate A Shiga. toxin effector polypeptide and/or Shiga toxin A Subunit polypeptide whether, naturally occurr ingor not, can be de- immunized by a method describe herein,d describe ind WO 2015/113005 and/or WO 2015/113007, and/or known to the skilled worker, wherein the resulting molecule retains one or more Shiga toxin A Subunit functions. 315. 315. 315. id="p-315" id="p-315" id="p-315" id="p-315" id="p-315" id="p-315" id="p-315" id="p-315" id="p-315" id="p-315"
[315] In certa embodimin ents, the Shiga toxin effector polypeptide of the present invention compris aes disruption of an endogenous epitope or epitope region, such as, e.g., a B-ceI Iand/o CD4+r T-cell epitope In. certain embodiments, the Shiga toxin effector polypeptide of the prese ntinvention compris aes disruption of at lea st one, endogenous, epitope regio descn ribe herein,d wherei then disrupti onreduces the antige nicand/o immunor geni potentic ofal the Shiga toxin effector polypept ide after administration of the polypeptide to a chordate and, wherein the Shiga toxin effector polypeptide is capable of exhibiting one or more Shiga toxin A Subuni t functions, such as, e.g,, a significa levent ofl Shiga toxin cytotoxicity. 316. 316. 316. id="p-316" id="p-316" id="p-316" id="p-316" id="p-316" id="p-316" id="p-316" id="p-316" id="p-316" id="p-316"
[316] The term "disrupted" or "disruption" as used herein with regard to an epitop e region refers to the deletion of at leas onet amino acid residue in an epitope region, inversion of two or more amino acid residues where at leas onet of the inverted amino acid residues is in an epitope region, inserti ofon at lea stone amino acid into an epitope region, and a substitution of at least one amino acid residue in an epitope region. An epitope regio disruptionn by mutati onincludes amino acid substituti ons wit hnon-standar aminod acid and/os non-nar tural amino acids Epitope. regions may alternatively be disrupt edby mutations comprisin theg modificatio of ann amino acid by the additi onof a covalently-li cheminkedcal structure which masks at leas onet amino acid in an epitope region, see, e.g. PEGylation (see Zhang C et al., BioDrugs 26; 209-15 (2012), smal moleculel adjuvants (Flowe D,r Expert OpinWO 2016/196344 PCT/US2016/034778 105 Drug Discov 1; 807-17 (2012), and site-specifi albuminac tion (Lim S et al., J Control Release 207-93 (2015)). 317. 317. 317. id="p-317" id="p-317" id="p-317" id="p-317" id="p-317" id="p-317" id="p-317" id="p-317" id="p-317" id="p-317"
[317] Certa epitopein regio nsand disruptions are indicate hereind by refere nceto specifi aminoc acid position ofs native Shiga toxin A Subunits provide ind the Sequence Listing, noting that natural occurringly Shiga toxin A Subunit smay comprise precurso formsr containing signal sequence ofs abou t22 amino acid ats thei amino-terr minals which are removed to produce matur Shigae toxin A. Subunits and are recognizabl to thee skilled worke r.Further cer, tai epitopen regio n disruptions are indicated herein by reference to specif icamino acids (e.g. S for a serine residue) natively prese ntat specif icpositions within native Shiga toxin A Subunits (e.g. S33 for the serine residue at position 33 from the amino-termin us) follow byed the amino acid with which that residue has been substitute in dthe particula mutatir onunder discussion (e.g. S33I represe ntsthe amino aci d substitut ionof isoleucine for serine at amino acid residue 33 from the amino- terminus). 318. 318. 318. id="p-318" id="p-318" id="p-318" id="p-318" id="p-318" id="p-318" id="p-318" id="p-318" id="p-318" id="p-318"
[318] In certa embodimein nts, the de-immunized, Shiga toxin effector polypept ide of the prese ntinvention compris aes disrupti onof at least one epitope region provided herei (seen e.g. Tables 1-7 and/or 12). In certa embodimein nts, the de- immunized, Shiga toxin effector polypeptide of the prese ntinvention compris aes disruption of at leas onet epitope region described in WO 2015/113 005 or WO 2015/113007. 319. 319. 319. id="p-319" id="p-319" id="p-319" id="p-319" id="p-319" id="p-319" id="p-319" id="p-319" id="p-319" id="p-319"
[319] In certa embodimein nts, the de-immunized, Shiga toxin effector polypept ide of the prese ntinvention compris ores consists essentially of a full-lengt Shigah toxin A Subunit (e.g .SLT-1A (SEQ ID NO: 1), Sb,A (SEQ ID NO:2), or SLT-2A (SEQ ID NO:3)) comprising at leas onet disruption of the amino aci dsequence selected from tiie group of natively positioned amino acids consisting of: 1-15 of SEQ ID NO:1 or SEQ ID NO:2: 3-14 of SEQ ID NO:3; 26 37 of SEQ ID NO:3: 27-37 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2: 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO: 1 or SEQ ID NO:2: 140-156 of SEQ ID NO:3: 179-190 of SEQ ID NO:1 or SEQ ID NO:2: 179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; 210-218 of SEQ IDNO:3; 240-258 of SEQ IDNO:3; 243-257 of SEQ ID NO: 1 or SEQ ID NO:2: 254-268 of SEQ ID NO: 1 or SEQ ID NO:2: 262-278 ofWO 2016/196344 PCT/US2016/034778 106 SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO: 1 or SEQ ID NO:2, or the equivalent position in a Shiga toxin A Subunit polypepti consde, erv ed Shiga toxin effector polypeptide sub-region, and/or non-nat ive,Shiga toxin effector polypept idesequence. 320. 320. 320. id="p-320" id="p-320" id="p-320" id="p-320" id="p-320" id="p-320" id="p-320" id="p-320" id="p-320" id="p-320"
[320] In certa emboin diments, the Shiga toxin effector polypeptide of the present invention compris ores consist esss entially of a truncat Shigaed toxin A Subunit.
Truncations of Shiga toxin A Subunits might resul int the deletion of an enti re epitope region(s without) affect ingShiga toxin effecto functr ion(s The). smalles t, Shiga toxin A Subunit fragment shown to exhibi signit fica enzymnt atic activity was a polypeptide composed of residues 75-247 of StxA (Al-Jaufy A et al.. Infect Immun 62: 956-60 (1994)). Truncating the carboxy-terminus of SLT-1 A, StxA, or SLT-2A to amino acids 1—251 remove twos predicted B-celi epitope regions two, predicted CD4 positive (CD4+) T-cell epitope ands, a predicte discd, ontinuous, B-cell epitope.
Truncating the amino-term inusof SLT-1 A, StxA, or SLT-2A to 75-293 removes at leas three,t predicte B-celld, epitope regions and three predicted CD4+ T-cell epitopes Trunc. ating both amino- and carboxy-termina of SLT-1A,ls StxA, or SLT- 2A to 75—251 deletes at leas five,t predicte B-celd, epitopel regions four; putati, ve, CD4+ T-cell epitope ands; one, predicte discontd, inuous, B-cell epitope. 321. 321. 321. id="p-321" id="p-321" id="p-321" id="p-321" id="p-321" id="p-321" id="p-321" id="p-321" id="p-321" id="p-321"
[321] In certa emboin diments, a Shiga toxin effector polypeptide of the invention may compris ore consist essentiall of ay fidl-lengt or truncath Shigaed toxin A Subunit wit hat leas onet mutation, e.g. deleti on,insertion, inversion, or substituti on, in a provide epitoped region. In certain furthe embodimer nts, the polypeptide s comprise a disruption whic hcompris aes deletion of at lea stone amino acid within tire epitope region. In certai furthern embodiments, the polypeptides comprise a disruption which compris anes inserti ofon at leas onet amino acid within the epitope region. In certain further embodiments, the polypepti descomprise a disruption which comprises an inversion of amino acids wherei, atn leas onet inverted ammo aci dis within the epitope region. In certa furthein embodr iment thes, polypepti des comprise a disruption which compris aes mutation, such as an ammo aci d substitut ionto a non-standar aminod acid or an amino acid with a chemical ly modified side chain. Numerous examples of single amino acid substitutions are provided in the Examples below. 322. 322. 322. id="p-322" id="p-322" id="p-322" id="p-322" id="p-322" id="p-322" id="p-322" id="p-322" id="p-322" id="p-322"
[322] In certa embodimein nts, the Shiga toxin effector polypepti desof the invention may comprise or consist essentiall of ay fidl-length or truncat Shigaed WO 2016/196344 PCT/US2016/034778 107 toxin A Subunit wit hone or more mutations as compared to the native sequence which compris ates least one amino acid substitution selected from tire group consisting of: A, G, V, L, I, P, C, M, F, S, D, N, Q, H, and K. In certa furtherin embodiments, the polypeptide may comprise or consis essentt iall of ay full-length or trunca tedShiga toxin A Subunit with a single mutation as compared to the native sequence wherein the substitution is selecte fromd the group consisting of: D to A, D to G, D to V, D to L, D to I, D to F, D to S, D to Q, E to A, E to G, E to V, E to L, E to I, E to F, E to S, E to Q, E to N, E to D, E to M, E to R, G to A, H to A, H to G, H to V, H to L, H to I, H to F, H to M, K to A, K to G, K to V, K to L, K to I, K to M, K to H, L to A, L to G, N to A, N to G, N to V, N to L, N to L N to F, P to A, P to G, P to F, R to A, R to G, R to V, R to L, R to I, R to F, R to M, R to Q, R to S, R to K, R to H, S to A, S to G, S to V, S to L, S to I, S to F, S to M, T to A, T to G, T to V, T to L, T to I, T to F, T to M, T to S, Y to A, Y to G, Y to V, Y to L, Y to I, Y to F, and Y to M. 323. 323. 323. id="p-323" id="p-323" id="p-323" id="p-323" id="p-323" id="p-323" id="p-323" id="p-323" id="p-323" id="p-323"
[323] In certa embodimein nts, the Shiga toxin effector polypepti desof the invention comprise or consist essentiall of ay full-length or truncat Shigaed toxin A Subunit wit hone or more mutations as compared to the native amino aci dresidue sequence which compris ates least one ammo acid substitution of an immunogenic residue and/o withinr an epitope region, wherein at leas onet substitution occurs at the native positly ioned group of amino acids selected from the group consisting of: I of SEQ ID NO: I or SEQ ID NO:2; 4 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 8 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID N():3; 11 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 33 of SEQ ID NO:1 or SEQ ID NO:2; 43 of SEQ ID NO:1 or SEQ ID NO:2: 44 of SEQ ID NO: 1 or SEQ ID NO:2; 45 of SEQ ID NO: 1 or SEQ ID NO:2; 46 of SEQ ID NO: I or SEQ ID NO:2; 47 of SEQ ID NO: 1 or SEQ ID NO:2; 48 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQ ID NO: 1 or SEQ ID NO:2: 50 of SEQ ID NO: 1 or SEQ ID NO:2; 51 of SEQ ID NO: 1 or SEQ ID NO:2; 53 of SEQ ID NO: 1 or SEQ ID NO:2; 54 of SEQ ID NO: 1 or SEQ ID NO:2; 55 of SEQ ID NO: 1 or SEQ ID NO:2: 56 of SEQ ID NO: I or SEQ ID N():2; 57 of SEQ ID NO: 1 or SEQ ID NO:2; 58 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3: 59 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 60 of SEQ ID NO: 1 or SEQ ID NO:2; 61 of SEQ ID NO: I or SEQ ID NO:2; 62 of SEQ ID NO;1 or SEQ ID NO:2; 84 of SEQ ID NO:1 or SEQ ID NO:2: 88 of SEQ ID NO:1 or SEQ ID NO:2; 94 ofWO 2016/196344 PCT/US2016/034778 108 SEQ ID NO; 1, SEQ ID NO:2, or SEQ ID NO:3; 96 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3: 104 of SEQ ID NO: 1 or SEQ ID NO:2; 105 of SEQ ID NO: 1 or SEQ ID NO:2; 107 of SEQ ID NO: 1 or SEQ ID NO:2: 108 of SEQ ID NO: 1 or SEQ ID NO:2; 109 of SEQ ID NO: i, SEQ ID NO:2, or SEQ ID NO:3; 110 of SEQ ID NO: 1 or SEQ ID NO:2; 111 of SEQ ID NO: 1 or SEQ ID NO:2; 112 of SEQ ID NO: I, SEQ ID NO:2, or SEQ ID NO:3; 141 of SEQ ID NO: 1 or SEQ ID NO:2: 147 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 154 of SEQ ID NO: 1 or SEQ ID NO:2; 179 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 180 of SEQ ID NO: I or SEQ ID XO 2: 181 of SEQ ID NO:1 or SEQ ID NO:2: 183 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 184 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 185 of SEQ ID NO: 1 or SEQ ID NO:2; 186 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 187 of SEQ ID NO: 1 or SEQ ID NO:2; 188 of SEQ ID NO: 1 or SEQ ID NO:2: 189 of SEQ ID NO: I or SEQ ID NO:2; 198 of SEQ ID NO: 1 or SEQ ID NO:2: 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2; 241 of SEQ ID NO:3; 242 of SEQ ID NO:i or SEQ ID NO:2; 247 of SEQ ID NO: I or SEQ ID NO:2; 247 of SEQ ID NO:3; 248 of SEQ ID NO: I or SEQ ID NO:2; 250 of SEQ ID NO:3: 251 of SEQ ID NO:1 or SEQ ID NO:2; 264 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 265 of SEQ ID NO: 1 or SEQ ID NO:2; and 286 of SEQ ID NO: 1 or SEQ ID NO:2. 324. 324. 324. id="p-324" id="p-324" id="p-324" id="p-324" id="p-324" id="p-324" id="p-324" id="p-324" id="p-324" id="p-324"
[324] In certain further embodiments, the Shiga toxin effector polypepti desof the invention comprise or consi stessentiall of ay full-length or trunca tedShiga toxin A Subunit with at lea stone substitution of an immunogenic residue and/o withinr an epitope region, wherein at leas onet amino acid substitution is to a non-conservat ive amino acid (see, e.g., Table C, infra) relat iveto a natively occurring amino aci d positioned at one of the followi nativeng positions: 1 of SEQ ID NO: 1 or SEQ ID NO:2: 4 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 8 of SEQ ID XO: L SEQ ID NO:2, or SEQ ID NO:3: 9 of SEQ ID XO: L SEQ ID NO:2, or SEQ ID NO:3; 11 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 33 of SEQ ID NO:1 or SEQ ID NO:2; 43 of SEQ ID NO: 1 or SEQ ID NO:2; 44 of SEQ ID NO: 1 or SEQ ID NO:2: 45 of SEQ ID NO: I or SEQ ID NO:2; 46 of SEQ ID NO: 1 or SEQ ID NO:2; 47 of SEQ ID NO: I or SEQ ID NO:2; 48 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQ ID NO: 1 or SEQ ID NO:2; 50 of SEQ ID NO: 1 or SEQ ID NO:2; 51 of SEQ ID NO: 1 or SEQ ID NO:2; 53 of SEQ ID NO: 1 or SEQ ID NO:2: 54 of SEQ ID NO: I or SEQ ID NO:2; 55 of SEQ ID NO:1 or SEQ ID NO:2;WO 2016/196344 PCT/US2016/034778 109 56 of SEQ ID NO: 1 or SEQ ID NO:2; 57 of SEQ ID NO: 1 or SEQ ID NO:2; 58 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO: A 59 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3: 60 of SEQ ID NO: 1 or SEQ ID NO:2; 61 of SEQ ID NO: 1 or SEQ ID NO:2; 62 of SEQ ID NO: 1 or SEQ ID NO:2; 84 of SEQ ID NO: 1 or SEQ ID NO:2; 88 of SEQ ID NO: 1 or SEQ ID NO:2; 94 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 96 of SEQ ID NO: 1, SEQ ID NO :2, or SEQ ID NO:3: 104 ofSEQ IDNO:lorSEQ ID NO:2; 105 of SEQ IDNO:1 or SEQ H) XO 2: 107 of SEQ ID NO: 1 or SEQ ID NO:2; 108 of SEQ ID NO: 1 or SEQ ID NO:2; 109 of SEQ ID NO: 1, SEQ IDNO:2, or SEQ IDNO:3; 110 ofSEQ IDNO:1 or SEQ IDNO:2; 111 ofSEQ IDNO:1 or SEQ IDNO:2; 112 ofSEQ H) NO: L SEQ IDNO:2, or SEQ IDNO:3; 141 ofSEQ ID NO:1 orSEQIDNO:2; 147 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 154 of SEQ ID NO: 1 or SEQ ID NO:2; 179 of SEQ ID NO:L SEQ IDNO:2, or SEQ ID NO:3; 180 ofSEQ IDNO:1 or SEQ IDNO:2; 181 of SEQ ID NO: 1 or SEQ ID NO:2; 183 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 184 ofSEQ IDNO:1, SEQ IDNO:2, or SEQ IDNO:3; 185 ofSEQ IDNO:1 or SEQ ID NO:2; 186 ofSEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 187 of SEQ IDNO:1 orSEQIDNO:2; 188 ofSEQ IDNO:1 or SEQ ID NO:2; 189 ofSEQ ID NO: 1 or SEQ ID NO:2; 198 of SEQ ID NO: 1 or SEQ ID NO:2; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2; 241 of SEQ ID NO:3; 242 of SEQ ID NO: 1 or SEQ ID NO:2: 247 of SEQ ID NO: 1 or SEQ ID NO:2; 247 of SEQ ID NOG: 248 of SEQ ID NO: 1 or SEQ ID NO:2; 250 of SEQ ID NO:3; 251 of SEQ ID NO: i or SEQ ID NOG: 264 of SEQ ID NO: 1, SEQ ID NOG, or SEQ ID NO:3; 265 ofSEQ ID NO:1 or SEQ ID NO:2; and 286 ofSEQ ID NO:1 or SEQ ID NOG.. 325. 325. 325. id="p-325" id="p-325" id="p-325" id="p-325" id="p-325" id="p-325" id="p-325" id="p-325" id="p-325" id="p-325"
[325] In certain further embodiments, the Shiga toxin effector polypepti desof the invention comprise or consist essentiall of a.y full-leng orth trunca tedShiga toxin, A Subunit with at leas onet amino acid substitution selected from the group consist ing of: KI to A, G, V, L, I, F, M and H; T4 to A, G, V, L, I, F, M, and S; D6 to A, G, V, L, I, F, S, and Q; S8 to A, G, V, I, L, F, and M; T8 to A, G, V, I, L, F, M, and S; T9 to A, G, V, I, L, F, M, and S; S9 to A, G, V, L, I, F, and M; KI 1 to A, G, V, L, I, F, M and H; T12 to A, G, V, I, L, F, M, and S; S33 to A, G, V, L, I, F, and M; S43 to A, G, V, L, I, F, and M; G44 to A and L; S45 to A, G, V, L, I, F, and M; T45 to A, G, V, L, I, F, and M; G46 to A and P; D47 to A, G, V, L, I, F, S, and Q; N48 to A, G, V, L, and M; L49 to A or G; F50; A51 to V; D53 to A, G, V, L, I, F, S, and Q; V54 to A, G, and L; R55 to A, G, V, L, 1, F, M, Q, S, K, and H; G56 to A and P; 157 WO 2016/196344 PCT/US2016/034778 110 to A, G, M, and F; L57 to A, G, M, and F; D58 to A, G, V, L, I, F, S, and Q; P59 to A, G, and F; E60 to A, G, V, L, I, F, S, Q, N, D, M, and R; E61 to A, G, V, L, I, F, S, Q, N, D, M, and R; G62 to A; D94 to A, G, V, L, I, F, S, and Q; R84 to A, G, V, L, I, F, M, Q, S, K, and H; V88 to A and G; 188 to A, G, and V; D94; S96 to A, G, V, I, L, F, and M; T104 to A, G, V, I, L, F, M, and S; A105 to L; T107 to A, G, V, I, L, F, M, and S; S107 to A, G, V, L, I, F, and M; L108 to A, G, and M; S109 to A, G, V, I, L, F, and M; T109 to A, G, V, I, L, F, M, and S; G110 to A; DI 11 to A, G, V, L, I, F, S, and Q; SI 12 to A, G, V, L, I, F, and M; D141 to A, G, V, L, I, F, S, and Q; G147 to A: V154 to A and G; R179 to A, G, V, L, I, F, M, Q, S, K, and H: T180 to A, G, V, L, I, F, M, and S; T181 to A, G, V, L, I, F, M, and S; DI83 to A, G, V, L, I, F, S, and Q; D184 to A, G, V, L, I, F, S, and Q; L185 to A, G, and V; S186 to A, G, V, I, L, F, and M; G187 to A; R188 to A, G, V, L, I, F, M, Q, S, K, and H; SI89 to A, G, V, I, L, F, and M; DI97 to A, G, V, L, I, F, S, and Q; DI98 to A, G, V, L, I, F, S, and Q; R204 to A, G, V, L, I, F, M, Q, S, K, and H; R205 to A, G, V, L, I, F, M, Q, S, K and H; C242 to A, G, V, and S; S247 to A, G, V, I, L, F, and M; ¥247 to A, G, V, L, I, F, and M; R248 to A, G, V, L, I, F, M, Q, S, K, and H; R250 to A, G, V, L, I, F, M, Q, S, K, and H; R251 to A, G, V, L, I, F, M, Q, S, K, and H; C262 to A, G, V, and S; D264 to A, G, V, L, I, F, S, and Q; G264 to A; and T286 to A, G, V, L, I, F, M, and S. 326. 326. 326. id="p-326" id="p-326" id="p-326" id="p-326" id="p-326" id="p-326" id="p-326" id="p-326" id="p-326" id="p-326"
[326] In certain further embodiments, the Shiga toxin effector polypepti desof the invention comprise or consi stessentiall of ay full-length or trunca tedShiga toxin A.
Subunit with at lea stone of the followi aminong acid substituti onsKIA, KIM, T4I, D6R, SSI, T8V, T9I, S9I, KHA, K11H, T12K, S33I, S33C, S43N, G44L, S45V, S451, T45V, T45I, G46P, D47M, D47G, N48V, N48F, L49A, F50T, A51V, D53A, D53N, D53G, V54L, V54I, R55A, R55V, R55L, G56P, I57F, I57M, D58A, D58V, D58F, P59A, P59F, E60I, E60T, E60R, E61A, E61V, E61L, G62A, R84A, V88A, D94A, S96I, T104N, A105L, T107P, L108M, S109V, T109V, G110A, D1HT, SI 12V, D141A, G147A, V154A R179A, T180G, ’FISH, D183A, D183G, D184A, D184A, D184F, L185V, L185D, S186A, S186F, G187A, G187T, R188A, R188L, S189A, D198A, R204A, R205A, C242S, S247I, Y247A, R248A, R250A, R251A, or D264A, G264A, T286A, and/or T286I. These epitope disrupting substitutions may be combined to form a de-immunize Shigad, toxin effecto polypeptr idewith multiple substitutions per epitope region and/or multiple epitope regions disrupted while still retaining Shiga toxin effector function. For example, substitutions at the WO 2016/196344 PCT/US2016/034778 111 natively positioned KIA, KIM, T4I, D6R, S8I, T8V, T9I, S9I, K11A, K11H, T12K, S331, S33C, S43N, G44L, S45V, S45I, T45V, T45I, G46P, D47M, D47G, N48V, N48F, L49A, F50T, A51V, D53A, D53N, D53G, V54L, V54I, R55A, R55V, R55L, G56P, I57F, I57M, D58A, D58V, D58F, P59A, P59F, E60I, E60T, E60R, E61A, E61V, E61L, G62A, R84A, V88A, D94A, S96I, T104N, A105L, T107P, L108M, S109V, T109V, G110A, D111T, S112V, D141A, G147A, V154A, R179A, T180G, T181I, D183A, D183G, D184A, D184A, D184F, L185V, L185D, S186A, S186F, G187A, G187T, R188A, R188L, S189A, D198A, R204A, R205A, C242S, S247I, Y247A, R2.48A, R250A, R251A, or D264A, G264A, T286A, and/o T286Ir may be combine d,wher posse ible w7,ith substituti onsat the natively positioned residues KIA, KIM, T4I, D6R, S8I, T8V, T9I, S9I, KI 1 A, KI 1H, T12K, S33I, S33C, S43N, G44L, S45V, S45I, T45V, T45I, G46P, D47M, D47G, N48V, N48F, L49A, F50T, A51V, D53A, D53N, D53G, V54L, V541, R55A, R55V, R55L, G56P, I57F, 15 7M, D58A, D58V, D58F, P59A, P59F, E60I, E60T, E60R E61A, E61V, E61L, G62A, R84A, V88A, D94A, S96I, T104N, A105L, T107P, L108M, S109V, T109V, G110A, Dll IT, SI 12V, D141A, G147A, V154A, R179A, T180G, T181I, D183A, D183G, D184A, D184A, D184F, L185V, L185D, S186A, S186F, G187A, G187T, R188A, R188L, S189A, D198A, R204A, R205A, C242S, S247I, Y247A, R248A, R250A, R25 LA, or D264A, G264A, T286A, and/or T286I to create de-immunize d, Shiga toxin effector polypepti desof the invention. 327. 327. 327. id="p-327" id="p-327" id="p-327" id="p-327" id="p-327" id="p-327" id="p-327" id="p-327" id="p-327" id="p-327"
[327] Any of the de-immunized, Shiga toxin effector polypeptide sub-regions and/o epitoper disrupting mutations described here inmay be used alone or in combination wit heach individua embodimel ofnt the prese ntinvention, including methods of the present invention.
B, Protease-Cleavage Resistant Shiga, Toxin A Subunit Effector Polypeptides 328. 328. 328. id="p-328" id="p-328" id="p-328" id="p-328" id="p-328" id="p-328" id="p-328" id="p-328" id="p-328" id="p-328"
[328] In certa emboin diments, the Shiga toxin effector polypept ideof the present invention comprises (1) a Shiga toxin Al fragment derived regio havingn a carboxy- terminus and (2) a disrupt edfurin-cleavage motif at the carboxy-terminus of the Shiga toxin Al fragment region. Improving the stabilit ofy connections between the Shiga toxin component and othe componentsr of cell-target molecules,ing e.g., cell- targeting binding regions can, improve their toxicit profiley afters administrat to ion organisms by reducin non-specifig toxicidec causeds by the breakdow ofn the connection and loss of cell-targeti suchng, as, e.g., as a resul oft proteolysis.WO 2016/196344 PCT/US2016/034778 112 329. 329. 329. id="p-329" id="p-329" id="p-329" id="p-329" id="p-329" id="p-329" id="p-329" id="p-329" id="p-329" id="p-329"
[329] Shiga toxin A Subunit sof members of the Shiga toxin family comprise a conserved, funn-cleavage site at the carboxy-termina of theirl Al fragment regio ns importa fornt Shiga toxin function. Furin-cleav siteage motifs and furin-cleava ge sites can be identifie byd the skilled worker using standard technique and/ors by using the information herein. 330. 330. 330. id="p-330" id="p-330" id="p-330" id="p-330" id="p-330" id="p-330" id="p-330" id="p-330" id="p-330" id="p-330"
[330] The model of Shiga toxin cytotoxi iscit thaty intracellula proteolytr ic process ingof Shiga toxin A Subunit sby furin in intoxicated cel lsis essential for 1) liberati ofon the Al fragment from the res oft the Shiga holotoxin, 2) escape of the Al fragment from the endoplasmic reticul umby exposing a hydrophobic domai inn the carboxy-terminus of the Al fragment, and 3) enzymat actic ivati ofon the Al fragment (see Johannes L, Romer W, Nat Rev Microbiol 8: 105-16 (2010)). Tire efficient liberat ionof the Shiga toxin Al fragment from the A2 fragment and the rest of the compone ntsof the Shiga holotoxin in the endoplasmic reticulum of intoxicat celed lsis essential for efficient intracellula routingr to the cytos ol,maxima l enzymat actic ivity effic, ient ribosome inactivat andion, achieving optimal cytotoxicity, i.e. comparable to a wild-type Shiga toxin (see e.g. WO 2015/191764 and references therein). 331. 331. 331. id="p-331" id="p-331" id="p-331" id="p-331" id="p-331" id="p-331" id="p-331" id="p-331" id="p-331" id="p-331"
[331] During Shiga toxin intoxicati theon, A Subuni tis proteolytically cleaved by furin at the carboxy' bond of a conserv arginineed residue (e.g. the arginine residue at position 251 in StxA and SLT-1A and tire arginine residue at position 250 in Stx2A and SLT-2A). Furin cleava ofge Shiga toxin A Subunit soccurs in endosoma and/ol r Golgi compartme nts.Furin is a. specialized serine endoprote whichase is expressed by a wide variety of cell types in, all human tissue examined,s and by most animal cell s.Furin cleave polypeptis descomprisin accg essible motif oftens center oned the minima l,dibasic, consens motifus R-x-(R/K/x)-R. The A Subunit sof members of the Shiga toxin family' comprise a conserved, surface-expose extended, loopd structure (e.g. 242-261 in StxA and SLT-1A, and 241-260 in SLT-2) wit ha conserv S-R/Y-x-ed x-R motif which is cleaved by' furin. The surfa ceexposed, extend looped structure positioned at amino acid residues 242-261 in StxA is require ford furin-induce clead vage of StxA, including feature flanks ing the minima l,funn-cleavage motif R-x-x-R. 332. 332. 332. id="p-332" id="p-332" id="p-332" id="p-332" id="p-332" id="p-332" id="p-332" id="p-332" id="p-332" id="p-332"
[332] Furin-cleav motifsage and furin-cleava sitege ins Shiga toxin A Subunit sand Shiga toxin effector polypepti descan be identifie byd the skilled worker using standar methd ods and/o byr using tire informati hereion n.Furin cleaves the WO 2016/196344 PCT/US2016/034778 113 minima l,consens motifus R-x-x-R (Schalken J et al., J Clin Invest 80: 1545-9 (1987): Bresnahan P et al., J Cell Biol 111; 2851-9 (1990); Matsuzaw Ka et al., J Biol Chem 265: 22075-8 (1990); Wise R et al., Proc Natl Acad Set USA 87: 9378-82 (1990); Molloy S et al., J Biol Chem 267: 16396-402 (1992)). Consiste witnt hthis , many furi ninhibitors comprise peptides comprisi ngthe motif R-x-x-R. An exampl e of a synthetic inhibitor of form is a molecule comprisin theg peptide R-V-K-R (SEQ ID NO:537) (Henrich S et al., Nat Struct Biol 10: 520-6 (2003)). In general, a peptide or protein comprisin a gsurface accessible, dibasi aminoc acid motif wit h two positively charged, amino acid seps ara tedby two amino acid residues may be predicted to be sensitive to forin-clea vagewith cleava occge urr ingat the carboxy bond of the last basi camino acid in the motif. 333. 333. 333. id="p-333" id="p-333" id="p-333" id="p-333" id="p-333" id="p-333" id="p-333" id="p-333" id="p-333" id="p-333"
[333] Consensus motif ins substrate cleaveds by fori haven been identifie withd some degree of specifici ty.A furin-cleava sitege motif has been described that compris aes region of twenty, continuous amino, acid residues which, can be label ed P14 throug P6h’ (Tian S et al., IntJMol Sci 12: 1060-5 (2011)) using the nomenclature described in Schechter I, Berge A,r, Biochem Biophys Res Commun 32: 898-902 (1968). According to this nomenclat theure, forin-clea vagesite is at the carboxy bond of the amino acid residue designat Pl,ed and the amino acid residues of the forin-clea vagemotif are numbered P2, P3, P4, etc .,in the direction going towar thed amino-termi fromnus this refere ncePl residue. Ilie amino acid residues of the motif going towa rdthe carboxy-termi fromnus the Pl refere nceresidue are numbered with the prime notat ionP2\ P3’, P4’, etc. Using this nomenclatu there, P6 to P2’ region delinea tiretes core substrat ofe the furin cleava motifge which is bound by the enzymat domaiic ofn form Hie. two flanking regions P14 to P7 and P3־ to P6’ are ofte richn in polar, amino acid residues to increas thee accessibility to the core furin cleavage site located between them. 334. 334. 334. id="p-334" id="p-334" id="p-334" id="p-334" id="p-334" id="p-334" id="p-334" id="p-334" id="p-334" id="p-334"
[334] A gener al,form-cleavage site is often describe byd the consensus motif R-x- x-R which corresponds to P4-P3-P2-P1; where "R" represents an arginine residue (see Table A, supra), a dash represe ntsa peptide bond, and a lowercas "x"e represents any amino acid residue. However othe, resr idues and positions may help to furthe defr ine furin-cleava motifsge A. slightly more refined forin-cleava site,ge consensus motif is ofte repon rte as dthe consens motifus R-x-[K/R]-R (wher a.e forwa slasrd "h/" mean "sor" and divides alternat aminoive acid residues at the same WO 2016/196344 PCT/US2016/034778 114 position) which, correspo tonds P4-P3-P2-P1, because it was observe thatd furin has a strong preference for cleaving substrat containies thisng motif. 335. 335. 335. id="p-335" id="p-335" id="p-335" id="p-335" id="p-335" id="p-335" id="p-335" id="p-335" id="p-335" id="p-335"
[335] In addition to the minimal fiirin-clea, vagesite R-x-x-R, a larger, fiirin- cleava motifge has been described with certa aminoin acid residue preferences at certain positions. By comparing various known furi nsubstrates, certain physicochem propertical haveies been characte rizedfor the amino acids residues in a amino acid resid ue long, fiirin-cleavage site motif. The P6 to P2/ region of the furin-cleava motifge delinea thetes core fiirin-cleavage site whic hphysically interacts wit hthe enzymati domainc of furin. The two flanking regions P14 to P7 and P3’ to P6’ are often hydrophilic being rich in polar, amino acid residues to increase the surface accessibility of the core furin-cleavage sit elocate betwd een them. 336. 336. 336. id="p-336" id="p-336" id="p-336" id="p-336" id="p-336" id="p-336" id="p-336" id="p-336" id="p-336" id="p-336"
[336] In general, the fiirin-clea vagemotif region from position P5 to Pl tends to comprise amino aci dresidues with a positive charge and/o highr isoelectric points.
In particula ther, Pl position, w'hich mark thes position of fiirin proteolys is is, generally occupie byd an arginine but other positively charged, amino acid residues may occur in this position. Positions P2 and P3 tend to be occupie byd flexible , amino acid residues, and in particular P2 tends to be occupie byd arginine, lysine, or sometimes by very small and flexible amino acid residues like glycine The. P4 position tends to be occupie byd positively charge aminod, acid residues in furi n substrates However. if ,the P4 position is occupie byd an aliphatic, amino acid residue, then the lack of a positive charged,ly functiona groupl can be compensated for by a positively charged residue located at position(s) P5 and/or P6. Positions Pl’ and P2’ are commonly occupied by aliphatic and/o hydrophobicr amino aci d residues with, the Pl’ position most commonly being occupied by a. serine. 337. 337. 337. id="p-337" id="p-337" id="p-337" id="p-337" id="p-337" id="p-337" id="p-337" id="p-337" id="p-337" id="p-337"
[337] The two, hydrophilic, flanking regions tend to be occupie byd amino aci d residues which are polar, hydrophili andc, have smaller amino acid functiona l groups however,; in certain verified fiirin substrate thes, flanking regio nsdo not contain any hydrophili ammoc, acid residues (see Tian S, Biochem Insights T. 9-20 (2009)). 338. 338. 338. id="p-338" id="p-338" id="p-338" id="p-338" id="p-338" id="p-338" id="p-338" id="p-338" id="p-338" id="p-338"
[338] lire twenty amino acid residue, fiirin-cleavage motif and fiirin-cleavage site found in native, Shiga toxin A Subunits at the junction between the Shiga toxin Al fragment and A2 fragment is well characteriz in certaed Shigain toxins. For example in StxA (SEQ ID NO:2) and SLT-1A (SEQ ID NO: 1), tins furin-cleavagemot ifis natively positioned from L238 to F257, and in SLT-2A (SEQ ID NO:3), this furin-cleava motige isf native positly ioned from V237 to Q256. Based on amino acid homology, experime nt,and/or furin-cleava assage ysdescribed herein, the skill edworker can identify furin-cleava motigefs in other native Shig, atoxin A Subunits or Shiga toxin effector polypeptides, wher thee motifs are actual furin­ cleavage motifs or are predict toed result in the product ionof Al and A2 fragment s after furin cleavage of those molecules within a eukaryot cellic . 339. 339. 339. id="p-339" id="p-339" id="p-339" id="p-339" id="p-339" id="p-339" id="p-339" id="p-339" id="p-339" id="p-339"
[339] In certai embn odiments the ,Shiga toxi effen ctor polypept ideof the present invention compris (1)es a Shiga toxin Al fragment derived polypept idehaving a , carboxy-term andinus (2) a disrupted furin-cleava motge ifat the carboxy-term inus of the Shiga toxin Al fragme derivednt polypepti de.The carboxy-term ofinus a Shig atoxin Al fragme derint ved polypeptide may be identifi byed the skill ed worker by using techniques known in the art, such as, e.g.״ by using protein sequence alignment softwa tore identify (i) a furin-cleavage moti consf erve withd a natural occuly rrin Shigag toxin, (ii) a surface expose d,extended loop conserved wit h a natural occurringly Shiga toxin and/or, (iii) a stretch of amino acid residues which are predomina ntlyhydrophobic (i.e. a hydrophob "patchic ") that may be recognized by the ERAD system. 340. 340. 340. id="p-340" id="p-340" id="p-340" id="p-340" id="p-340" id="p-340" id="p-340" id="p-340" id="p-340" id="p-340"
[340] A protease-cleava resistgeant, Shiga toxi effen ctor polypept ideof the present invention (1) may be complete lackly ing any furin-cleava motge ifat a carboxy­ terminus of its Shiga toxin Al fragme regionnt and/or (2) comprise a disrupted furin-cleavage moti atf the carboxy-termi ofnus it sShig atoxin Al fragment region and/or region derived from the carboxy-term ofinus a Shiga toxi Aln fragment. A disruption of a furin-cleava motge ifincludes various alterati toons an amino acid residue in the furin-cleavage motif, such as, e.g., a post-transla modiftionication(s), an alterati ofon one or more atom ins an amino aci dfunctional group, the additi onof one or more atom tos an amino acid functional group, the association to a non- proteinaceou moiets y(ies) and/or, the linkage to an amino acid residue, peptide, polypept idesuch as result ingin a branche proteid nace structous ure. 341. 341. 341. id="p-341" id="p-341" id="p-341" id="p-341" id="p-341" id="p-341" id="p-341" id="p-341" id="p-341" id="p-341"
[341] Protease-cleavage resistant, Shiga toxi effen ctor polypepti desmay be creat ed from a Shiga toxin effector polypepti and/orde Shiga toxi An Subunit polypepti de, whether natura occully rrin or gnot, using a method described herei n,described in WO 2015/191764, and/or known to the skilled worker, wherein the result ing molecule still retai nsone or more Shiga toxin A Subunit functions. -115-WO 2016/196344 PCT/US2016/034778 116 342. 342. 342. id="p-342" id="p-342" id="p-342" id="p-342" id="p-342" id="p-342" id="p-342" id="p-342" id="p-342" id="p-342"
[342] For purposes of the prese ntinvention wit hrega rdto a furin-cleava sitege or funn-cleav motif,age the term "disruption־’ or "disrupte" refersd to an altera tion from the natural occlyurr ingfurin-cleava sitege and/o furin-clr eavage motif, such as, e.g., a mutation, that results in a reduction in furin-cleavage proximal to the carboxy- termin ofus a Shiga toxin Al fragment region, or identifia regble ion derive therd eof, as compared to the funn-cleavage of a wild-type Shiga toxin A Subunit or a polypeptide derived from a wild-type Shiga toxin A Subunit comprisi ngonly wild- type polypeptide sequenc esAn. altera tionto an amino acid residue in Ilie furin- cleava motifge includes a mutati onin the furin-cleavage motif, such as, e.g., a deletion, insertion, inversion, substitution, and/or carboxy-termi trunnalcati ofon the furin-cleava motif,ge as wel asl a post-translati modifion cat ion,such as, e.g., as a result of glycosylati albumion, nation, and the like which involve conjugating or linking a molecule to the functiona groupl of an amino acid residue. Becaus thee fiirin-clea vagemotif is compris edof about twenty, amino acid residues in, theory', alterations, modificati ons,mutations deletions,, insertions, and/o truncar tions involving one or more amino acid residues of any one of these twent positionsy might resul int a reduction of furin-cleava sensgeitivi (Tiaty n S et al., Set Rep 2: 261 (2012)). Ure disruption of a fiirin-cleavage site and/or furin-cleavage motif may or may not increase resista nceto cleava byge oilier protease suchs, as, e.g., trypsin and extracellular proteas commes on m the vascular system of mammals. Hie effects of a given disruption to cleava sensge itivi ofty a given protease may be teste byd the skilled worker using technique knowns in the art. 343. 343. 343. id="p-343" id="p-343" id="p-343" id="p-343" id="p-343" id="p-343" id="p-343" id="p-343" id="p-343" id="p-343"
[343] For purposes of the prese ntinvention, a "disrupted furin-cleava motifge’ is fiirin-clea vagemotif comprising an altera tionto one or more amino acid residues derived from the 20 amino acid residue regio repn resenti a conseng rved, furin- cleava motifge found in native Shiga, toxin A Subunit sat the junction between the Shiga toxin Al fragment and A2 fragment regions and positioned such that furin cleava ofge a Shiga toxin, A Subunit results in the producti ofon the A1 and A2 fragments; wherei then disrupted fiirin-cleavage motif exhibits reduc edfurin cleava inge an experimental reproduciblely way as compared to a refere nce molecule comprisin a gwild-type, Shiga toxin Al fragment region fused to a carboxy-terminal polypeptide of a size large enoug toh monitor furin cleava usingge the appropri ateassay known to the skilled worke and/orr describe hereid n.WO 2016/196344 PCT/US2016/034778 117 344. 344. 344. id="p-344" id="p-344" id="p-344" id="p-344" id="p-344" id="p-344" id="p-344" id="p-344" id="p-344" id="p-344"
[344] Example ofs types of mutations whic hcan disrupt a furin-cleavage site and furin-cleava motifge are ammo acid residue deletions, insertions, truncations, inversions and/or, substitutions, including substitutions wit hnon-standar aminod acid and/ors non-natur aminoal acids In. addition, fiirin-clea vagesites and furin- cleava motifge cans be disrupt edby mutations comprising the modification of an amino acid by the additi onof a covalently-li strucnkedture which masks at lea stone amino acid in the site or motif, such as, e.g., as a resul oft PEGylation, the coupling of sma llmolecule adjuvants, and/or site-specifi albuminac tion. 345. 345. 345. id="p-345" id="p-345" id="p-345" id="p-345" id="p-345" id="p-345" id="p-345" id="p-345" id="p-345" id="p-345"
[345] If a fiirin-cleavage motif has been disrupt edby mutation and/o ther presence of non-natur aminoal acid residues cer, tai disruptedn furin-cleavage motif mays not be easily recogniza asble being related to any fiirin-cleavage motif; however, the carboxy-termi ofnus the Shiga toxin A1 fragment derive regd ion will be recognizable and will define where tire furin-cleavage motif would be located wer e it not disrupted. For example, a disrupte fiirin-cleavaged motif may compris lesse than the twenty, ammo acid residues of the fiirin-cleavage motif due to a carboxy- terminal truncat asion compared to a Shiga toxin A Subunit and/o Shigar toxin Al fragment. 346. 346. 346. id="p-346" id="p-346" id="p-346" id="p-346" id="p-346" id="p-346" id="p-346" id="p-346" id="p-346" id="p-346"
[346] In certain embodiments, the Shiga toxin effector polypeptide of the present invention comprises (1) a Shiga toxin Ai fragment derived polypeptide having a carboxy-termi andnus (2) a disrupted furin-cleavage motif at the carboxy-terminus of the Shiga toxin Al fragment polypept ideregion; wherein the Shiga toxin effector polypept ide(and any cell-targetin molecg ule comprising it) is more fiirin-cleavage resist antas compar edto a refere ncemolecule such, as, e.g., a wild-type Shiga toxin polypeptide comprising the carboxy-terminus of an Al fragment and/o ther conserved, fiirin-clea vagemotif between Al and A2 fragments. For example, a reduction in furin cleavage of one molecule compared to a refere ncemolecule may be determine usingd an in vitro, furin-cleavage assa descy ribe ind tire Example s below, conducte usingd the same conditions, and then performi ang quantitat ofion the band density of any fragmen resultts ingfrom cleava toge quantitatively measure in change in furi ncleavage. 347. 347. 347. id="p-347" id="p-347" id="p-347" id="p-347" id="p-347" id="p-347" id="p-347" id="p-347" id="p-347" id="p-347"
[347] In certa embodimein nts, the Shiga toxin effector polypeptide is more resistan to furit n-cleava in vitroge and/or in vivo as compared to a wild-type, Shiga toxin A Subunit.WO 2016/196344 PCT/US2016/034778 118 348. 348. 348. id="p-348" id="p-348" id="p-348" id="p-348" id="p-348" id="p-348" id="p-348" id="p-348" id="p-348" id="p-348"
[348] In general, the protease-cle avagesensitivi ofty a cell-targeti moleculeng of tiie prese ntinvention is tested by comparing it to the same molecule having its furin- cleava resge ista Shigant, toxin effecto polypeptider replace witd ha wild-type, Shiga toxin effector polypeptide comprising a Shiga toxin Al fragment. In certain embodiments, the molecules of the prese ntinvention comprisi nga disrupted furin- cleava motifge exhibits a reduction in in vitro furin cleavage of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% or greate comparr toed a reference molecule comprising a wild-type, Shiga toxin A1 fragment fused at its carboxy-termi to nusa peptide or polypeptide, such as, e.g., the refere ncemolecule SLT-lA-WT::scFv-l described in Example 2, below. 349. 349. 349. id="p-349" id="p-349" id="p-349" id="p-349" id="p-349" id="p-349" id="p-349" id="p-349" id="p-349" id="p-349"
[349] Several furin-cleava motifge disruptions have been describe d.For example, mutating the two conserv argied nines to alanines in the minimal R-x-x-R moti f complete blockedly processing by furm and/or furin-like proteas (seees e.g Duda A et al., J Virology 13865-70 (2004)). Because the furin-cleava sitege motif is compris edof about twenty amino acid residues in, theor certy, ain mutations involving one or more of any one of these twenty, amino acid residue positions might abolish furin cleavage or reduce furin cleavage efficiency (see e.g. Tian S et al7&״/?ep2: 261 (2012)). 350. 350. 350. id="p-350" id="p-350" id="p-350" id="p-350" id="p-350" id="p-350" id="p-350" id="p-350" id="p-350" id="p-350"
[350] In certa embodimein nts, the molecules of the prese ntinvention compris ae Shiga toxin effector polypeptide derived from at lea stone A Subunit of a member of the Shiga toxin family wherein the Shiga, toxin effector polypeptide compris aes disruption in one or more amino acids derived from the conserved, highly accessibl proteasee, -clea sensvageitive loop of Shiga toxin A Subunits. For example, in StxA and SLT-1A, this highly accessibl protease, e-sens loopitive is natively positioned from ammo acid residues 242 to 261, and in SLT-2A, this conserv looped is native positly ioned from amino acid residues 241 to 260. Base d on polypept idesequence homology, the skilled worker can identif thisy conserved, highly accessibl loope structur in eother Shiga toxin A Subunits. Certa mutatin ions to the amino aci dresidues in this loop can reduce the accessibi lityof certai aminon acid residues within the loop to proteolytic cleavage and this might reduce furin- cleava sensge itivity. 351. 351. 351. id="p-351" id="p-351" id="p-351" id="p-351" id="p-351" id="p-351" id="p-351" id="p-351" id="p-351" id="p-351"
[351] In certain embodiments, a molecul ofe the present invention compris aes Shiga toxin effector polypeptide comprisin a gdisrupt edfurin-cleavage motif comprising a mutation in the surface-expose protead, sesensitive loop conserve dWO 2016/196344 PCT/US2016/034778 119 among Shiga toxin A Subunits. In certain furthe embodimer nts, a molecule of the present invention comprises a Shiga toxin effector polypept idecomprisi nga disrupted furin-cleavage motif comprisin a gmutati onin this protease-sens loopitive of Shiga toxin A Subunits the, mutation which reduce the surface accessibility of certain amino acid residues within the loop such that furin-cleava sensgeitivi isty reduced. 352. 352. 352. id="p-352" id="p-352" id="p-352" id="p-352" id="p-352" id="p-352" id="p-352" id="p-352" id="p-352" id="p-352"
[352] In certa emboin diments, the disrupted furin-cleava motifge of a Shiga, toxin effector polypeptide of the prese ntinvention compris aes disruption in terms of existenc position,e, or functional group of one or both of the consens aminous acid residues Pl and P4, such as, e.g., the amino acid residues in positions I and 4 of the minimal furin-cleava motifge R/Y-x-x-R. For example, mutating one or both of the two arginine residues in the minimal furin, consens siteus R-x-x-R to alanine will disrup at furin-cleava motifge and prevent furin-cleavage at that site. Similarly, amino acid resi due substituti onsof one or both of the arginine residues in the minimal furin-cleava motifge R-x-x-R to any non-conservat amiiveno aci dresidue known to die skilled worker will reduced the furin-cleavage sensitivit of ythe motif.
In particular amino, aci dresidue substitutions of arginine to any non-basic amino acid residue which lack as positive charge, such as, e.g.. A, G, P, S, T, D, E, Q, N, C, I, L, M, V, F, W, and Y, will result in a disrupted furin-cleava motif.ge 353. 353. 353. id="p-353" id="p-353" id="p-353" id="p-353" id="p-353" id="p-353" id="p-353" id="p-353" id="p-353" id="p-353"
[353] In certa emboin diments, the disrupt edfurin-cleava motifge of a Shiga toxin effector polypeptide of the prese ntinvention compris aes disruption in the spacin g between the consensus amino aci dresidues P4 and Pl in term ofs the number of intervening amino acid residues being other than two, and, thus, changing eithe P4r and/o Plr into a differ entposition and eliminati theng P4 and/o Plr designation s.
For example, deletions wdthi then furin-cleava motifge of the minimal furin-cleavage site or the core, furin-cleava motifge will reduce the furin-cleavage sensitivi ofty the furin-cleava motif.ge 354. 354. 354. id="p-354" id="p-354" id="p-354" id="p-354" id="p-354" id="p-354" id="p-354" id="p-354" id="p-354" id="p-354"
[354] In certa emboin diments, the disrupted furin-cleava motifge compris onees or more amino acid residue substitutions as compared, to a wild-type, Shiga toxin A Subunit. In certain further embodiments, the disrupt edfurin-cleavage motif compris onees or more ammo acid residue substitutions within the minimal furin- cleava sitege R/Y-x-x-R, such as, e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptide thes, native positily oned amino acid residue R248 substituted with any non-positively charged, amino acid residue and/or R251 substitute withd WO 2016/196344 PCT/US2016/034778 120 any non-positively charged, amino acid residue; and for SLT-2A derived Shiga toxin effector polypeptide tires, natively positioned amino aci dresidue ¥247 substituted with any non-positively charged, amino aci dresidue and/or R250 substituted with any non-positively charged, amino acid residue. 355. 355. 355. id="p-355" id="p-355" id="p-355" id="p-355" id="p-355" id="p-355" id="p-355" id="p-355" id="p-355" id="p-355"
[355] In certa emboin diments, the disrupted furin-cleavage motif compris anes un- disrupted, minimal furin-cleavage site R/Y-x-x-R but instead compris aes disrupted flanking region, such as, e.g., amino aci dresidue substitutions in one or more amino acid residues in the furin-cleava motifge flanking regions natively position at, e.g., 241-247 and/or 252-259. In certai furthen embodimer nts, the disrupt edfuri n cleava motifge compri ses a substitution of one or m ore of the amino aci dresidues located in the Pl-P6 region of the furin-cleava motif;ge mutati ngPl’ to a bulky amino acid such, as, e.g., R, W, Y, F, and H; and mutating P2’ to a polar and hydrophilic amino acid residue; and substituting one or more of the amino aci d residues locat ined the P1 ’-P6 ’ regio ofn the furin-cleavage motif with one or more bulky and hydrophobic amino acid residues 356. 356. 356. id="p-356" id="p-356" id="p-356" id="p-356" id="p-356" id="p-356" id="p-356" id="p-356" id="p-356" id="p-356"
[356] In certa embodimein nts, the disruption of the furin-cleavage motif comprises a deleti on,insertion, inversion, and/o mutatir onof at leas onet amino acid residue withi then furin-cleavage motif. In certain embodiments, a protease-cle avage resistant, Shiga toxin effector polypeptide of the present invention may comprise a disruption of the ammo acid sequence natively positioned at 249-251 of the A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) or Shiga toxin (SEQ ID NO:2), or at 247-250 of the A Subunit of Shiga-like toxin 2 (SEQ ID NO:3) or the equivalent position in a conserve Shigad toxin effector polypeptide and/or non-native Shiga toxin effector polypeptide sequence In. certai furthern embodiment prots, eas e- cleava resge ista Shigant, toxin effecto polypeptidesr compris ae disruption which compris aes deletion of at leas onet amino acid within the furin-cleava motifge In. certain furthe embodr iment proteas, se-cleava resistgeant, Shiga toxin effector polypepti descompr sei a disruption which compris anes insertion of at leas onet amino acid within the protease-cleava motifge region. In certa furthein r embodiments, the protease-cleava resistgeant, Shiga toxin effector polypepti des comprise a disrupti onwhich compris anes inversion of ammo acids, wherein at least one inverted amino aci dis within the protea motifse region. In certain furthe r embodiments, the protease-cleava resistgeant, Shiga toxin effector polypepti des comprise a disrupti onwhich compris aes mutation, such as an ammo aci dWO 2016/196344 PCT/US2016/034778 121 substitut ionto a non-standar aminod acid or an amino acid with a chemical ly modified side chain. Examples of single ammo acid substitutions are provid edin the Example belows . 357. 357. 357. id="p-357" id="p-357" id="p-357" id="p-357" id="p-357" id="p-357" id="p-357" id="p-357" id="p-357" id="p-357"
[357] In certain embodiments of the molecules of the prese ntinvention, the disrupt edfurin-cleavage motif compris thees deletion of nine, ten, eleven, or more of the carboxy-termina ammlo acid residues within the furm-cleavage motif. In thes e embodiments, the disrupted furin-cleava motifge will not compris ae furin-cleavage site or a minimal furin-cleava motif.ge In othe wordsr cer, tai embodimn ents lack a furm-cleavage site at the carboxy-terminus of the Al fragment region. 358. 358. 358. id="p-358" id="p-358" id="p-358" id="p-358" id="p-358" id="p-358" id="p-358" id="p-358" id="p-358" id="p-358"
[358] In certa emboin diments, the disrupt edfurin-cleava motifge compris bothes an amino aci dresidue deletion and an amino acid residue substitution as compared to a wild-type, Shiga toxin A Subunit. In certai furthen embodr iment thes, disrupted furin-cleavage motif compris onees or more amino acid residue deletions and substitutions within the minimal furin-cleavage site R/Y-x-x-R, such as, e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptides the natively, positioned amino acid residue R248 substituted with any non-positively charged, amino aci d residue and/o R25r 1 substitute withd any non-positively charged, amino acid residue; and for SLT-2A derived Shiga toxin effector polypeptide thes, natively positioned amino acid residue Y247 substitute withd any non-positively charge d, amino acid residue and/or R250 substituted with any non-positively charge aminod, aci dresidue. 359. 359. 359. id="p-359" id="p-359" id="p-359" id="p-359" id="p-359" id="p-359" id="p-359" id="p-359" id="p-359" id="p-359"
[359] In certa emboin diments, the disrupted furin-cleava motifge compris anes amino acid residue deletion and an amino acid residue substitution as well as a carboxy-termina truncationl as compared to a wild-type Shiga, toxin A Subunit. In certain further embodiment thes, disrupted furin-cleava motifge compris onees or more amino acid residue deletions and substitutions within the minimal furin- cleava sitege R/Y-x-x-R, such as, e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptide thes, native positily oned amino acid residue R248 substituted wit hany non-positively charged, amino acid residue and/or R251 substitute withd any non-positively charged, amino acid residue; and for SLT-2A derived Shiga toxin effector polypeptide tires, natively positioned amino aci dresidue ¥247 substituted with any non-positively charged, amino acid residue and/or R250 substitute withd any non-positively charged, amino acid residue.WO 2016/196344 PCT/US2016/034778 122 360. 360. 360. id="p-360" id="p-360" id="p-360" id="p-360" id="p-360" id="p-360" id="p-360" id="p-360" id="p-360" id="p-360"
[360] In certain further embodiments, the disrupt edfurin-cleavage motif comprise s both an amino acid substitution within tire minimal furin-cleavage site R/Y-x-x-R and a carboxy-termina truncatil ason compared to a wild-type, Shiga toxin A Subunit, such as, e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptide truncationss, ending at the natively amino acid position 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, or great ander comprising die native positily oned amino acid residue R248 and/or R251 substituted with any non-positively charge d, amino acid residue wher appropriatee and ;for SLT-2A derived Shiga toxin effector polypeptide truncas, tions ending at the natively amino aci dposition 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, or great ander comprisin theg natively positioned amino acid residue Y247 and/o R250r substituted with any non-positively charged , amino acid residue where appropriate. 361. 361. 361. id="p-361" id="p-361" id="p-361" id="p-361" id="p-361" id="p-361" id="p-361" id="p-361" id="p-361" id="p-361"
[361] In certa emboin diments, the disrupt edfurin-cleava motifge compri ses an insertion of one or more ammo acid residues as compar toed a wild-type, Shiga toxin A Subunit as long as the insert amied no residue( doess) not create a de novo furin- cleava site.ge In certai embodimen nts, the inserti ofon one or more amino aci d residues disrupts the natural spacing between the arginine residues in the minima l, furin-cleavage site R/Y-x-x-R, such as, e.g., StxA and SLT-1A derived polypepti des comprising an inserti ofon one or more amino acid residues at 249 or 250 and thus between R248 and R251; or SLT-2A derived polypeptide comprs isin ang insertion of one or more amino acid residues at 248 or 249 and thus between Y247 and R250. 362. 362. 362. id="p-362" id="p-362" id="p-362" id="p-362" id="p-362" id="p-362" id="p-362" id="p-362" id="p-362" id="p-362"
[362] In certa emboin diments, the disrupt edfurin-cleava motifge compris bothes an amino acid residue inserti andon a carboxy-termina truncal tion as compared to a wild-type, Shiga toxin A Subunit. In certain embodiments, the disrupt edfurin- cleava motifge compris bothes an amino aci dresidue insertion and an amino aci d residue substitution as compar toed a wild-type, Shiga toxin A Subunit. In certai n embodiments, the disrupted furin-cleava motifge compris bothes an amino aci d residue insertion and an amino aci dresidue deletion as compared to a wild-type, Shiga toxin A Subunit.WO 2016/196344 PCT/US2016/034778 123 363. 363. 363. id="p-363" id="p-363" id="p-363" id="p-363" id="p-363" id="p-363" id="p-363" id="p-363" id="p-363" id="p-363"
[363] In certa emboin diments, the disrupt edfurin-cleavage motif compris anes amino acid residue deletio ann, ammo acid residue inserti on,and an ammo aci d residue substitution as compared to a wild-type, Shiga toxin A Subunit. 364. 364. 364. id="p-364" id="p-364" id="p-364" id="p-364" id="p-364" id="p-364" id="p-364" id="p-364" id="p-364" id="p-364"
[364] In certain embodiments, the disrupt edfurin-cleava motifge compris anes amino acid residue deletio insertion,n, substitution, and carboxy-termina truncationl as compared to a wild-type, Shiga toxin A Subunit. 365. 365. 365. id="p-365" id="p-365" id="p-365" id="p-365" id="p-365" id="p-365" id="p-365" id="p-365" id="p-365" id="p-365"
[365] In certa embodimein nts, the Shiga toxin effecto polypeptider comprisin a g disrupt edfurin-cleavage motif is direct fusedly by a peptide bond to a molecul ar moiety comprisi ngan amino acid peptide,, and/or polypeptide wherei then fused structure involves a single continuous, polypeptide. In these fusion embodiments, the amino acid sequence followi theng disrupt edfurin-cleavage motif should not creat a ede novo, furin-cleava sitege at the fusion junction. 366. 366. 366. id="p-366" id="p-366" id="p-366" id="p-366" id="p-366" id="p-366" id="p-366" id="p-366" id="p-366" id="p-366"
[366] Any of tire above protease-cleava resistgeant, Shiga toxi effen ctor polypeptide sub-regions and/or disrupte furin-d cieavage motifs may be used alone or in combination wit heach individua embodl iment of the prese ntinvention, includi ng methods of the present invention.
C. T-Cell Hyper-Immunize Shigad, Toxin A Subunit Effect Polypeptidesor 367. 367. 367. id="p-367" id="p-367" id="p-367" id="p-367" id="p-367" id="p-367" id="p-367" id="p-367" id="p-367" id="p-367"
[367] In certa emboin diments, the Shiga toxin effector polypeptide of the present invention comprises an embedded or insert epitopeed -pept ide.In certain further embodiments, the epitope-pepti isde a heterologous T-ce,ll epitope-pept ide,such as, e.g., an epitope considere heted rologous to Shiga toxin A Subunits. In certa in furthe embodimer nts, the epitope-pepti isde a CD8+ T-cell epitope In. certa in furthe embodr iment thes, CD 8+ T-cell epitope-pepti hasde a bindin gaffinity to a MHC class I molecule characteri byzed a dissociation consta (Kntd) of 104־ molar or less and/o ther result ingMHC class I-epitope-pept compleide hasx a bindin gaffinit y to a T-cell receptor (TCR) characteriz by eda dissociation consta (Kntd) of 104־ molar or less. 368. 368. 368. id="p-368" id="p-368" id="p-368" id="p-368" id="p-368" id="p-368" id="p-368" id="p-368" id="p-368" id="p-368"
[368] In certa emboin diments, the Shiga toxin effector polypeptide of the present invention comprises an embedded or inserte heted, rologous T-ce,il epitope, such as, e.g., a huma nCD8+ T-cell epitope In. certa furthein embodr iment thes, heterologous T-ce,ll epitope is embedde ord inserted so as to disrupt an endogenous epitope or epitope region (e.g. a B-cell epitope and/o CD4+r T-ceil epitope) identifia inble a naturally occurring Shiga toxin polypept ideor parental Shiga toxineffector polypept idefrom whic hthe Shig atoxin effector polypeptide of the present invention is derived. 369. 369. 369. id="p-369" id="p-369" id="p-369" id="p-369" id="p-369" id="p-369" id="p-369" id="p-369" id="p-369" id="p-369"
[369] For certai embodimn ents of the present invention, the Shiga toxi effectorn polypept ide(and any cell-targeti molngecul compe rising it) is CD8+ T-cel hyperl ­ immunized, such as, e.g., as compared to a wild-type Shiga toxi polypen ptide The.
CD8+ T-cel hyper-imml unized, Shig atoxin effector polypeptide of thes present invention eac hcomprise an embedde ord inserted T-cel epitl ope-pepti de.Hype­r immunized, Shig atoxi effectn polypor epti descan be create fromd Shiga toxin effect polypeor ptide and/ors Shig atoxi An Subunit polypeptides, whether natura lly occurrin or gnot, using a method described herei n,described in WO 2015/113007, and/or known to the skille worked whereinr, the result ingmolecule still retains one or more Shig atoxin A Subunit functions. 370. 370. 370. id="p-370" id="p-370" id="p-370" id="p-370" id="p-370" id="p-370" id="p-370" id="p-370" id="p-370" id="p-370"
[370] For purposes of the claime inventd ion, a T-cell epitope is a molecula r structure whic his comprised by an antigenic peptide and can be represented by a linear, amino acid sequence. Commonly, T-cell epitopes are peptides of size sof eight to eleven amino acid residues (Townsend A, Bodmer H, Annu Rev Immunol I: 601-24 (1989)); however, certain T-cell epitope-pept idehaves lengt thaths are smalle thanr eight or larg thaner eleven amino acids long (see e.g. Livingstone A, Fathman C, Annu Rev Immunol 5: 477-501 (1987); Gree nK et al., Eur J Immunol 34: 2510-9 (2004)). In certain embodiments, the embedded or inserted epitope is at leas sevet namino acid residues in length. In certai embodimn ents, the embedded or inserted epitope is bound by a TCR wit ha binding affini characty teri byzed a Kd less than 10 mM (e.g. 1-100 pM) as calcula usingted the formul ina Stone J et al., Immunology 126: 165-76 (2009). However, it should be noted that the binding affini witty hin a given range between the MHC-epitope and TCR may not correlat e with antigenici and/orty immunogenicit (seey e.g. Al-Ramadi B et ah, J Immunol 155: 662-73 (1995)), such as due to factors like MHC-peptide-TCR complex stabilit MHC-pepy, tide densit andy MHC-independent functions of TCR cofact ors such as CD8 (Bake Br et al., Immunity 13: 475-84 (2000); Hornell T et al., J Immunol 170: 4506-14 (2003); Woolridge L et al., J Immunol 171: 6650-60 (2003)). 371. 371. 371. id="p-371" id="p-371" id="p-371" id="p-371" id="p-371" id="p-371" id="p-371" id="p-371" id="p-371" id="p-371"
[371] A heterologou T-cells, epitope is an epitope not alrea presentdy in a wil­d type Shiga toxi An Subunit; a natural occurly ring Shiga toxi An Subunit; and/or a parental, Shiga toxin effector polypepti usedde as a source polypepti forde -124-WO 2016/196344 PCT/US2016/034778 125 modification by a method describe herein,d describe ind WO 2015/113007, and/or known to the skilled worker. 372. 372. 372. id="p-372" id="p-372" id="p-372" id="p-372" id="p-372" id="p-372" id="p-372" id="p-372" id="p-372" id="p-372"
[372] A heterologous, T-cell epitope-pept mayide be incorporated into a source polypeptide via numerous methods known to the skille worker,d including, e.g., the processes of creating one or more amino acid substitutions within the sourc e polypepti de,fusing one or more amino acids to the sourc polype epti de,inserting one or more amino acid intos the sourc polype epti linkingde, a peptide to the source polypepti and/ode, ar combination of the aforementioned process es.The result of such a method is the creati ofon a modified variant of the sourc polypeptidee which compris onees or more embedded or inserted hete, rologous T-ce,ll epitope-peptides. 373. 373. 373. id="p-373" id="p-373" id="p-373" id="p-373" id="p-373" id="p-373" id="p-373" id="p-373" id="p-373" id="p-373"
[373] T-cell epitopes may be chosen or derive frdom a number of sourc moleculee s for use in the prese ntinvention. T-cell epitopes may be creat ored derive frdom various naturally occurring protei ns.T-cell epitopes may be create ord derive frdom various naturally occurring proteins foreign to mammals, such as, e.g., proteins of microorganism T-ces.ll epitopes may be creat ored derived from mutated human proteins and/o humanr protei nsaberrant exprely ss byed maligna humant ncells T-. cell epitop esmay be synthetica creatlly ored derived from synthetic molecules (see e.g., Carbone F et al., J Exp Med 167: 1767-9 (1988); Del Vai M et al., J Virol 65: 3641-6 (1991); Appella E et al., Biomed Pept Proteins Nucleic Acids 1: 17784־ (1995); Perez S etal Cancer., 116: 2071-80 (2010)). 374. 374. 374. id="p-374" id="p-374" id="p-374" id="p-374" id="p-374" id="p-374" id="p-374" id="p-374" id="p-374" id="p-374"
[374] Although any T-cell epitope-pept iside contemplat as beined g used as a heterologous T-ce,ll epitope of the prese ntinvention, certa epitopin esmay be selected based on desirable properti es.One objective of the prese ntinvention is to creat CD8+e T-cell hyper-immunized, Shiga toxin effector polypeptides for administrati to vertebraon meantes, ing that the heterologous, T-cell epitope is highly immunogeni andc can elicit robust immune response in svivo when display ed complexed wit ha MHC clas Is molecule on the surfac ofe a cell In. certa in embodiments, the Shiga toxin effector polypeptide of the present invention compris onees or more, embedde ord inserte heted, rologous T-ce,ll epitopes which are CD8+ T-cell epitopes A. Shiga toxin effecto polypeptider of the present invention that comprises a heterologous, CD8+ T-cell epitope is considere a CD8+d T-cell hyper-immunized, Shiga toxin effector polypeptide. 375. 375. 375. id="p-375" id="p-375" id="p-375" id="p-375" id="p-375" id="p-375" id="p-375" id="p-375" id="p-375" id="p-375"
[375] T-cell epitope compone ntsof the prese ntinvention may be chosen or derived from a number of sourc moleculee alreas knowndy to be capable of eliciting a WO 2016/196344 PCT/US2016/034778 126 vertebr immuneate response. T-cell epitopes may be derived from various naturally occurring proteins foreign to vertebrates such ,as, e.g., proteins of pathogenic microorganisms and non-self cance, antiger ns.In particular infect, ious microorganisms may contain numerous proteins with known antigenic and/o r immunogeni propertic es.Further, infectious microorganisms may contain numerous proteins with known antigenic and/or immunogeni sub-regionsc or epitopes. 376. 376. 376. id="p-376" id="p-376" id="p-376" id="p-376" id="p-376" id="p-376" id="p-376" id="p-376" id="p-376" id="p-376"
[376] For example, the protei nsof intracellula pathogensr with mammalia hostsn are sourc esfor T-cell epitopes There. are numerous intracell pathogens,ular such as viruses, bacter fungi,ia, and single-cell eukaryote withs, well-studie antid genic proteins or peptides T-ce. ll epitopes can be selected or identifi fromed human viruses or othe intracr ellula pathogenr suchs, as, e.g., bacteria like mycobacter ium, fungi like toxoplasmae, and protists like trypanosomes. 377. 377. 377. id="p-377" id="p-377" id="p-377" id="p-377" id="p-377" id="p-377" id="p-377" id="p-377" id="p-377" id="p-377"
[377] For example, there are many immunogenic, vira peptidel components of vira l proteins from, viruses that are infectious to humans Numerous,. human T-cell epitopes have been mappe dto peptid eswithin proteins from influenza. A viruses , such as peptides in the proteins HA glycoprotei FEIns7, S139/1, CH65, C05, hemagglutin 1 (HAI), hemagglutin 2 (HA2)in , nonstruct proteinural 1 and. 2 (NS1 and NS 2), matrix protei 1n and 2 (Ml and M2), nucleoprotein (NP), neuraminid ase (NA)), and many of thes peptidese have been shown to elic humanit immune responses such, as by using ex vivo assay. Similarly, numerou humans, T-cell epitopes have been mappe dto peptide compone ntsof proteins from human cytomegalovirus (HCMV),es such as peptides in the protei pp65ns (UL83), ULI 28- 131, immediate-ear 1 (IE-ly1; ULI23), glycoprote B, teguin ment proteins and, many of these peptides have been shown to elicit human immune respons es,such as by using ex vivo assays. 378. 378. 378. id="p-378" id="p-378" id="p-378" id="p-378" id="p-378" id="p-378" id="p-378" id="p-378" id="p-378" id="p-378"
[378] Another exampl ise there are many immunogenic, cance antigensr in humans The. CD8+ T-cell epitopes of cance and/orr tumor cell antigens can be identifi ed,by' the skilled worke usingr techniques known in the art, such as, e.g., differentia genomil cs, different protial eomic imms, unoproteom predicics, tion then validation, and genetic approaches like reverse-genetic transfec (seetion e.g., Admon A et al., Moi Cell Proteomics 2: 388-98 (2003); Purce llA, Gorma J,n Mol Cell Proteomics 3: 193-208 (2004); Comber J, Philip R, Ther Adv Vaccines 2: 77-89 (2014)). There are many antigenic and/o immunor geni T-cec ll epitopes already identifie ord predicted to occur in human cance and/orr tumor cells. For example, T- WO 2016/196344 PCT/US2016/034778 127 cell epitop eshave been predicted in huma nproteins commonly mutat edor overexpres insed neoplas cellstic such, as, e.g., ALK, CEA, N-acetylglucosaminyl- transfe raseV (GnT-V), HCA587, HER-2/neu, MAGE, Melan-A/MAR.T-1, MUC-1, p53, and TRAG-3 (see e.g., van der Brugge Pn et al., Science 254: 1643-7 (1991); Kawakami ¥ et al., J Exp Med 180: 347-52 (1994); Fisk B et al., J Exp Med 181: 2109-17 (1995); GuillouxYetal.,JExpMe 183: 1173d (1996); Skippe rJ et al., J Exp Med 183: 527 (1996); BrossartP etaL, 93: 4309-17 (1999); Kawashima I etaL, Cancer Res 59: 431-5 (1999); Papadopoul Kos et al., Clin Cancer Res 5: 2089-93 (1999); Zhu B et al., Clin Cancer Res 9: 1850-7 (2003); Li B et al., Clin Exp Immunol 140: 310-9 (2005); Art-Tahar K et al., Ini J Cancer 118: 688-95 (2006); Akiyam Ya et al., Cancer Immunol Immunother 61: 2311 -9 (2012)). In addition, syn the tic varia ntsof T-cell epitopes from huma ncance celr lshave been creat (seede e.g., Lazoura E, Apostolopoul V, osCurrMed Chern 12: 629-39 (2005); Dona t- Casass usC et al., J Med Chern 50: 1598-609 (2007)). 379. 379. 379. id="p-379" id="p-379" id="p-379" id="p-379" id="p-379" id="p-379" id="p-379" id="p-379" id="p-379" id="p-379"
[379] AVhil eany T-cell epitope may be used in the polypepti desand molecules of tiie prese ntinvention, certain T-cell epitopes may be preferr baseded on their known and/o empirr ical detely rmined character istForics example,. in many species, the MHC alleles in its genome encode multiple MHC-I molecul variar ants Becaus. e MHC cla ssI protei polymorphin smscan affect antigen-MHC class I complex recognition by CD8+ T-cells, T-cell epitopes may be chosen for use in tire present invention based on knowledge about certa MHCin cla ssI polymorphi and/orsms the ability of certain antigen-MHC class I complexe to sbe recogniz byed T-cells having differ entgenotypes. 380. 380. 380. id="p-380" id="p-380" id="p-380" id="p-380" id="p-380" id="p-380" id="p-380" id="p-380" id="p-380" id="p-380"
[380] There are well-define peptided -epitope thats are known to be immunogenic, MHC cla ssI restricte and/ord, matched with a specifi humac nleukocyte antigen (HLA) variant(s) For. applications in humans or involving human targ cellset , HLA-class !-restric epittedopes can be selected or identif iedby the skilled work er using standar techniquesd known in the art. The ability of peptides to bind to human MHC class I molecule cans be used to predi ctthe immunogeni potentiac of putativel T-cell epitopes The. ability of peptid esto bind to human MHC cla ssI molecules can be scor edusing software tools. T-cell epitopes may be chosen for use as a heterologous T-ce,ll epitope component of the prese ntinvention base don the peptide selectivity of the HLA variants encode byd the alle lesmore prevalent in certain human populations. For example, the huma npopulation is polymorphi forc WO 2016/196344 PCT/US2016/034778 128 tiie alpha chain of MHC class I molecule dues to the varied alleles of the HLA genes from individual to individual. In certai T-cen ll epitopes may be more efficiently presente byd a specific HLA. molecule, such as, e.g., the commonly occurring HLA variant encodeds by the HLA-A allele groups HLA-A2 and HLA-A3. 381. 381. 381. id="p-381" id="p-381" id="p-381" id="p-381" id="p-381" id="p-381" id="p-381" id="p-381" id="p-381" id="p-381"
[381] When choosing T-cell epitopes for use as a heterologous T-ce,ll epitop e component of the prese ntinvention, multiple factors may be considere thatd can influenc epite ope generation and transpor to receptivet MHC class I molecule suchs, as, e.g., the presence and epitope specific ityof the following factors in the target cell: proteasome, ERAAP/ERAP1, tapasin, and TAPs. 382. 382. 382. id="p-382" id="p-382" id="p-382" id="p-382" id="p-382" id="p-382" id="p-382" id="p-382" id="p-382" id="p-382"
[382] When choosing T-cell epitopes for use as a heterologous T-ce,ll epitop e component of the prese ntinvention, epitope may be selected which best matc theh MHC class I molecules present in the cell-type or cell populations to be targeted.
Differe MHCnt cla ss1 molecule exhibits preferentia bindingl to particular peptide sequence ands, particular peptide-MHC clas Is varia complexesnt are specifically recogniz byed the t-ce llreceptors (TCRs) of effecto T-cellsr Hie. skilled worker can use knowledge about MHC class I molecule specificit andies TCR specificit toies optimize the selectio ofn heterologous T-ce,ll epitopes used in the present invention. 383. 383. 383. id="p-383" id="p-383" id="p-383" id="p-383" id="p-383" id="p-383" id="p-383" id="p-383" id="p-383" id="p-383"
[383] In addition, multiple, immunogenic, T-cell epitopes for MHC class I presentation may be embedded in the same Shiga toxin effector polypept ideof the present invention, such as, e.g., for use in the targe teddelivery of a plurality of T- cell epitop essimultaneously. An example of a cell-targeti molecng ule of the prese nt invention comprisin multipleg CD8+, T-cell epitopes is SEQ ID NO:26. 384. 384. 384. id="p-384" id="p-384" id="p-384" id="p-384" id="p-384" id="p-384" id="p-384" id="p-384" id="p-384" id="p-384"
[384] Any of the protease-cle avageresistant, Shiga toxin effecto polypeptider sub- regions and/or disrupt edfurm-cleav motifsage described herei mayn be used alone or in combinat ionwith each individual embodiment of the prese ntinvention, including methods of the prese ntinvention.
IL The Gener alStructures of the Cell-Target Molecing ules of the Invention 385. 385. 385. id="p-385" id="p-385" id="p-385" id="p-385" id="p-385" id="p-385" id="p-385" id="p-385" id="p-385" id="p-385"
[385] The Shiga toxin effector polypepti desof the prese ntinvention provide robust and powerful scaffolds for engineerin novel,g cell-targeti molecules.ng The associated of cell-targetin bindingg regions with Shiga toxin effector polypepti desof the prese ntinvention enables the engineering of therapeutic and diagnos tic molecules wit hdesirabl chae racter istsuchics as,, e.g., de-immunization, potent WO 2016/196344 PCT/US2016/034778 129 cytotoxici efficty, ient intracell routinular T-celg, hyper-iml munizati molecon, ular stabilit andy, in vivo tolerability at high dosages. 386. 386. 386. id="p-386" id="p-386" id="p-386" id="p-386" id="p-386" id="p-386" id="p-386" id="p-386" id="p-386" id="p-386"
[386] The present invention provides various cell-targeti moleculesng each, comprising (1) a cell-targeting, binding regio andn (2) a Shiga toxin effector polypept ideof the present invention. The Shiga toxin effecto polypepr tides of the present invention may be associa tedwith and/o coupledr to various dive, rse, cell - targeting compone nts(e.g. a molecul moietyar and/o agentr to) create cell-targetin g molecules of the present invention. A cell-target moleingcule of the present invention comprises (1) a bindin regg ion capable of specificall bindiny gan extracellular part of a targe biomolecult ande (2) a Shiga toxin effector polypeptide regio comprn isi nga Shiga toxin, effecto polypeptider of the prese ntinvention. 387. 387. 387. id="p-387" id="p-387" id="p-387" id="p-387" id="p-387" id="p-387" id="p-387" id="p-387" id="p-387" id="p-387"
[387] The Shiga toxin effector polypepti desof the prese ntinvention may be linked to one or more cell-targe bindinting, gregions that mediat cell-e target viaing binding specifici toty extracel partlular ofs targe biomolect ules, such as, e.g., a targe t biomolecul physice ally couple tod a cellular surface of a cell One. non-limiti ng example of a cell-targeti molecng ule of the prese ntinvention is a Shiga toxin effector polypeptide of the prese ntinvention fused to a proteinace s, cell-ou targe ting, bindin gregion, such as, e.g., an immunoglobulin- bindintype gregion.
A. Binding Regions 388. 388. 388. id="p-388" id="p-388" id="p-388" id="p-388" id="p-388" id="p-388" id="p-388" id="p-388" id="p-388" id="p-388"
[388] In certa embodimein nts, a binding region of a cell-targetin molegcule of the present invention is a cell-targeting component, such as, e.g., a. domai n,molecular moiet y,or agent, capable of bindin gspecifica tolly an extracellular part of a target biomolecul (e.g.e an extracellular target biomolecule with) high affinity. There are numerous types of bindin gregio nsknown to skilled worker or which may be discovere by dthe skill edworker using techniques known in the art. For exampl e, any cell-targeti componentng that exhibits the requisite binding characterist ics describe hereid mayn be used as the binding regio inn certain embodiments of the cell-target moleculeing ofs the present invention. 389. 389. 389. id="p-389" id="p-389" id="p-389" id="p-389" id="p-389" id="p-389" id="p-389" id="p-389" id="p-389" id="p-389"
[389] An extracellular part of a targe biomt olec ulerefe rsto a portion of its structure expose tod the extracel lularenvironmen whent the molecule is physicall y couple tod a cel l,such as, e.g., when the targe biomolt ecul is expre esse atd a cellular surfa ceby the cel l.In this context, expose tod the extracel lularenvironmen meanst that part of the targ biomoleculet is acce essible by, e.g., an antibody or at leas at WO 2016/196344 PCT/US2016/034778 130 binding moiety smaller than an antibody such as a single-doma antiboin dydomai n,a nanobody, a heavy-chain antibody domain derived from camelids or cartilaginous fishes a, single-chain variable fragment, or any numbe rof engineered alternat ive scaffolds to immunoglobulins (see below) The. exposure to the extracellula r environmen of ort accessibility to a part of targ biomoleculet physice all coupledy to a cell may be empirical detely rmine by dthe skilled worker using methods well known in the art. 390. 390. 390. id="p-390" id="p-390" id="p-390" id="p-390" id="p-390" id="p-390" id="p-390" id="p-390" id="p-390" id="p-390"
[390] A bindin gregio ofn a cell-targeti molecng ule of the prese ntinvention may be, e.g., a ligand, peptide, immunoglobulin-type bindin gregion, monoclo nal antibody, engineer antibodyed derivat ive,or engineer alternated toive antibodies. 391. 391. 391. id="p-391" id="p-391" id="p-391" id="p-391" id="p-391" id="p-391" id="p-391" id="p-391" id="p-391" id="p-391"
[391] In certain embodiments, the binding regio ofn a cell-targetin molegcule of the prese ntinvention is a proteinace rnoietyous capable of bindin gspecifica tolly an extracellular part of target biomolecul withe high affinit Ay. binding region of a cell-target moleingcule of the prese ntinvention may comprise one or more various peptidic or polypeptide moieties such, as randomly genera tedpeptide sequences, naturally occurri ligang nds or derivatives thereof, immunoglobuli deriven domaid ns, synthetica engineerlly scaed ffolds as alternatives to immunoglobulin domains, and the like (see e.g., WO 2005/092917; WO 2007/033497; Cheung M et al., Mol Cancer 9: 28 (2010); US 2013/0196928; WO 2014/164693; WO 2015/113005; WO 2015/113007; WO 2015/138452; WO 2015/191764). In certain embodiments, a cell-target moleculeing of the prese ntinvention compris aes bindin regiog n comprising one or more polypepti descapable of selective andly specifica bindinglly an extracel lulartarge biomolecule.t 392. 392. 392. id="p-392" id="p-392" id="p-392" id="p-392" id="p-392" id="p-392" id="p-392" id="p-392" id="p-392" id="p-392"
[392] There are numerous binding regions known in the art that are useful for targeting molecules to specific cell-ty pesvia. thei bindinr charactg erist suchics, as certain ligands monoclonal, antibodie engineers, antibodyed derivati ves,and engineer alted erna tiveto antibodies s. 393. 393. 393. id="p-393" id="p-393" id="p-393" id="p-393" id="p-393" id="p-393" id="p-393" id="p-393" id="p-393" id="p-393"
[393] According to one specif icbut non-limiti aspngect, the binding region of a cell-target moleculeing of the prese ntinvention compris aes natural occurrly ing ligand or derivati thereofve that retains binding functionalit to any extracellula r target biomolecule, commonly a cell surfa cereceptor For. example, variou s cytokines, growth factors and, hormon knownes in the art may be used to target the cell-target moleculeing of the present invention to the cell-surfa ofce specif iccell types expressing a cognate cytoki recne eptor, growth factor receptor or hormo, nereceptor. Certa non-limin iting examples of ligands include (alternati namesve are indicate in dparentheses) angiogenin B-cell, activating factors (BAFFs, APRIL), colony stimulati factorsng (CSFs), epiderm growthal factors (EGFs), fibrobla st growth factors (FGFs), vascul endothear liagrowtl facth ors (VEGFs), insulin-li ke growth factors (IGFs), interferons, interleukins (such as IL-2, IL-6, and IL-23), nerve growt factorsh (NGFs), platel derivedet growth factors, transformi growthng factors (TGFs), and tumor necrosis factors (TNFs). 394. 394. 394. id="p-394" id="p-394" id="p-394" id="p-394" id="p-394" id="p-394" id="p-394" id="p-394" id="p-394" id="p-394"
[394] According to certa otherin embodiment of sthe cell-targeti molengcules of the prese ntinvention, the binding region comprises a syntheti ligandc capable of binding an extracell targetular biomolecule (see e.g. Liang S et al., J Mol Med 84: 764-73 (2006); Ahmed S et al., Anal Chern 82: 7533-41 (2010); Kaur K et al., Methods Mol Biol 1248: 239-47 (2015)). 395. 395. 395. id="p-395" id="p-395" id="p-395" id="p-395" id="p-395" id="p-395" id="p-395" id="p-395" id="p-395" id="p-395"
[395] In certai embodimn ents, the binding region comprises a peptidomimet ic, such as, e.g., an AApeptide, gamma-AApepti and/orde, sulfono-y-AApeptide (see e.g., Pilsl L, Reise rO, Amino Acids 41: 709-18 (2011); Akram O et al., Mol Cancer Res 12: 967-78 (2014); Wu H et al., Chemistry 21: 2501-7 (2015); Teng P et al., Chemistry 2016 Mar 4)). 396. 396. 396. id="p-396" id="p-396" id="p-396" id="p-396" id="p-396" id="p-396" id="p-396" id="p-396" id="p-396" id="p-396"
[396] According to one specific, but non-limiting aspect, the binding region may compri sean immunoglobulin-t bindiypeng region The. term "immunoglobulin-t ype binding region" as used herei refersn to a polypeptide region capable of binding one or more target biomolecules such, as an antigen or epitope. Binding regions may be functionall definey byd thei abir lit toy bind to target molecules Imm. unoglobuli n- type binding region ares commonly derive fromd antibody or antibody-like structures; however, alternat scaffolive fromds other source ares contempla ted withi then scop eof the term. 397. 397. 397. id="p-397" id="p-397" id="p-397" id="p-397" id="p-397" id="p-397" id="p-397" id="p-397" id="p-397" id="p-397"
[397] Immunoglobulin (Ig) protei havens a structural domain known as an Ig domai n.Ig domains range in length from about 70-110 amino acid residues and possess a characteri Ig-fold,stic in which typicall 7 toy 9 antipara betalle strandl s arrange into two beta sheets which form a sandwich-l strucike ture. The Ig fold is stabilized by hydrophob aminoic acid interactions on inne rsurfaces of the sandwic h and highly conserved disulfide bonds between cysteine residues in the strands. Ig domai nsmay be variable (IgV or V-set), constant (IgC or C-set) or intermedi (Iglate or I-set). Some Ig domai nsmay be associated wit ha complement aritdetermy ini ng region (CDR), also called a "complement determary inin region,g " which is -131-WO 2016/196344 PCT/US2016/034778 132 importa fornt the specificity of antibodies bindin tog thei epitopesr Ig-. like domains are also found in non-immunoglo bulinproteins and are classifi oned that basis as members of the Ig superfamily of protein Thes. HUGO Gene Nomenclat ure Committe (HGNC)e provide a slist of members of the Ig-like domain containing family. 398. 398. 398. id="p-398" id="p-398" id="p-398" id="p-398" id="p-398" id="p-398" id="p-398" id="p-398" id="p-398" id="p-398"
[398] An immunoglobulin-t bindingype region may be a polypeptide sequence of an antibody or antigen-binding fragment thereof wherein the amino acid sequence has been varie fromd that of a native antibody or an Ig-like domai ofn a non- immunoglobulin protein, for exampl bye molecul enginear erin or gselection by libra ryscreening. Because of the relevance of recombinant DNA techniques and tn vitro librar־ scry eenin ing the generation of immunoglobulin- bindintype gregions , antibodies can be redesigne to dobtain desired characterist suchics, as smaller size, cell entry, or other improveme fornts in vivo andJot therapeutic applications. The possible variatio arens many and may range from, the changing of just one amino aci dto the complete redesign of, for example, a variable region. Typically, changes in die variable region will be made in order to improve the antigen-binding character istimproveics, variable region stability, or reduce the potentia forl immunogeni responc ses. 399. 399. 399. id="p-399" id="p-399" id="p-399" id="p-399" id="p-399" id="p-399" id="p-399" id="p-399" id="p-399" id="p-399"
[399] There are numerous immunoglobulin-type binding regions contemplat as ed compone ntsof the present invention. In certai embodn iment thes, immunoglobulin- type bindin gregion is derived from an immunoglobulin binding region, such as an antibody paratope capabl ofe bindin gan extracellular target biomolecule. In certa in other embodiments, the immunoglobulin-type binding regio comprisn anes engineered polypept idenot derived from any immunoglobulin domain but which functions like an immunoglobulin bindin regg ion by providing high-affin bindingity to an extracellular targe biomolet cule. This engineer polypeptideed may optionally include polypeptide scaffold comprisings or consisting essentia oflly complementa ry determin regiing ons from immunoglobulin as dess crib edherein. 400. 400. 400. id="p-400" id="p-400" id="p-400" id="p-400" id="p-400" id="p-400" id="p-400" id="p-400" id="p-400" id="p-400"
[400] There are also numerous binding regions in the prior art that are usefill for targeti polypepting desto specif iccell-ty viapes their high-affinity binding character isticIn certas. embodimein nts, the binding region of the cell-targeting molecule of the prese ntinvention is selected from the group which includes autonomous Vh domains, single-doma antiin body domains (sdAbs), heavy-chain antibody domai nsderived from camelids (VhH fragments or Vh domai fragments),n WO 2016/196344 PCT/US2016/034778 133 heavy-chain antibody domains derived from camel VidhH fragments or Vh domain fragments, heavy-chain antibody domains derived from cartilaginou fishess , immunoglobulin new antigen recepto (IgNARsrs ), Vnar fragments, single-chai n variable (scFv) fragments, nanobodies, Fd fragmen consts ist ingof the heavy chain and ChI domains, single chai Fnv-Ch3 minibodies dimeri, Cch2 domain fragments (Ch2D), Fc antige bindingn domains (Fcabs), isolate complementad determiningry region 3 (CDR3) fragments, constra inedframewor regiok 3,n CDR3, framewor k regio 4n (FR3-CDR3-FR4) polypeptides sma, ll modular immunophannaceutical (SMTP) domains, scFv-Fc fusions, multimerizing scFv fragments (diabodies, triabodies, tetrabodies), disulfide stabiliz antibodyed variable (Fv) fragments, disulfid stabie lize antiged n-bindi (Fab)ng fragmen consists ting of the Vl, Vh, Cl and ChI domains, bivale nanobodint bivalees, minint bodie bivales, F(abnt ’)2 fragmen (Fabts dimers), bispecific tandem VhH fragments bispe, cif ictandem scFv fragments, bispecific nanobodies bispec, ific minibodies and, any genetical ly manipula tedcounterpart of thes foregoing that reta itsin paratope and binding function (see War dE et al., Nature 341: 544-6 (1989); Davies J, Riechmann L, Biotechnology (NY) 13: 475-9 (1995); Reiter Y et al., Mol Biol 290: 685-98 (1999); Riechma nnL, Muyldermans S, J Immunol Methods 231: 25-38 (1999); Tanha J et al., J Immunol Methods 263: 97-109 (2002); Vranke Wn et al., Biochemistry 41: 8570-9 (2002); Jespers L et al., J Mol Biol 337: 893-903 (2004); Jespers L et al., Nat Biotechnot M; 1161-5 (2004): To R et al., J Biol Chern. 280: 41395-403 (2005); Saerens D et al., Gurr Opin Pharmacol 8: 600-8 (2008); Dimitrov D, MAbs 1: 26-8 (2009); Weiner L, Cell 148: 1081-4 (2012); Ahmad Z et al., Clin Dev Immunol 2012: 980250 (2012)). 401. 401. 401. id="p-401" id="p-401" id="p-401" id="p-401" id="p-401" id="p-401" id="p-401" id="p-401" id="p-401" id="p-401"
[401] There are a variety of bindin regionsg comprisin polypeptidesg derived from the consta regionsnt of immunoglobuli suchns, as, e.g., engineer dimeried Fcc domains, monomeric Fes (mFcs) ,scFv-Fc s,VhH-Fcs, Ch2 domains, monome ric Ch3s domains (mCH3s) ,synthetica reprllyogramm immunoglobulied domainsn , and/o hybridr fusions of immunoglobulin domains wit hligan ds(Hofe Tr et al., Proc Natl Acad Set U. S. A. 105: 12451-6 (2008); Xiao J et al., 3 Am Chern Soc 131: 13616-13618 (2009); Xiao X et al., Biochem Biophys Res Commun 387: 387-92 (2009); Wozniak-Knopp G et al., Protein Eng Des Sei 23 289-97 (2010); Gong R et al., PLoS ONE 7: 642288 (2012); Wozniak-Knopp G et al., PLoS ONE 7: 630083 (2012); Ying T et al., J Biol Chern 287: 19399-408 (2012); Ying T et al., J BiolWO 2016/196344 PCT/US2016/034778 134 Chem 288: 25154-64 (2013); Chiang M et al., J Am Chem Soc 136: 3370-3 (2014); Rader C, Trends Biotechnol 32: 186-97 (2014); Ying T et al., Biochirmca Biophys Acta 1844: 1977-82 (2014)). 402. 402. 402. id="p-402" id="p-402" id="p-402" id="p-402" id="p-402" id="p-402" id="p-402" id="p-402" id="p-402" id="p-402"
[402] In accordance with certa othein embodimer nts, the binding region comprises an engineere altd, ernati scaffoldve to immunoglobul domaiin ns. Engineer ed alternat scaiveffolds are known in the art whic hexhibit similar functional characteristics to immunoglobulin-deri structures,ved such as high-affinity and specif icbindin gof targe biomolect ules, and may provide improve chard acter toist ics certain immunoglobulin domains, such as, e.g., great staer bili orty reduced immunogenic ity.General altely, rnat sciveaffolds to immunoglobulins are les thans kilodaltons consist, of a single polypeptide chain, lack cysteine residues and, exhibit relative highly thermodynamic stability. 403. 403. 403. id="p-403" id="p-403" id="p-403" id="p-403" id="p-403" id="p-403" id="p-403" id="p-403" id="p-403" id="p-403"
[403] For certain embodimen ofts the cell-targeti molecng ules of the present invention, the binding region compris anes alternat scaffoldive selected from the group which includes autonomous Vh domains, single-domain antibody domain s (sdAbs), heavy-chain antibody domains derived from camelids (VhH fragmen orts Vh domai fragmn ents) heavy-cha, antiin body domain derives fromd camelid VhH fragmen orts Vh domain fragments, heavy-chain antibody domains derived from cartilaginous fishe s,immunoglobulin new antigen receptor (IgNARs)s ,VNAR fragments, single-chai varin able (scFv) fragments, nanobodies, Fd fragments consisting of the heavy chain and ChI domains, permutate Fvsd (pFv), singl chaine Fv-Ch3 minibodies dimeri, Cch2 domai frn agments (Ch2D), Fc antigen binding domains (Fcabs) isola, ted complementa determiningry region 3 (CDR3) fragments, constra inedframework region 3, CDR3, framework region 4 (FR3-CDR3-FR4) polypeptide smals, modularl immunopharmaceut (SMTPica)l domains, scFv-Fc fusions, multimerizi scFvng fragmen (diabodies,ts triabodies, tetrabodies), disulfi de stabilized antibody variable (Fv) fragments, disulfide stabiliz antigen-bindinged (Fab) fragmen consistingts of the Vl, Vh, Cl and ChI domains, bivalent nanobodies, bivalent minibodies bivale, F(abnt ’)2 fragmen (Fabts dimers) bispec, ific tandem VhH fragments, bispecif ictandem scFv fragments, bispecific nanobodi bispecifes, ic minibodies and, any genetically manipulat counted erpa ofrts the foregoi thatng retains its bindin funcg tionali (Worty nA, Pluckthun A, J Mol Biol 305: 989-1010 (2001); Xu L et al., Chem Biol 9: 933-42 (2002); Wikman M et al., Protein Eng Des Sei 17: 455-62 (2004); Binz H et al., Nat Biotechnol 23: 1257-68 (2005); Hey T et WO 2016/196344 PCT/US2016/034778 135 al., Trends Biotechnol 23 :514-522 (2005); Holliger P, Hudson P, Nat Biotechnol 23: 1126-36 (2005); Gill D, Daml N,e Curr Opin Biotech 17: 653-8 (2006); Koide X, Koide S, Methods Mol Biol 352: 95-109 (2007); Byla P et al., J Biol Chern 285: 12096 (2010); Zoller F et al.,Molecules 16: 2467-85 (2011); Alfarano P et al., Protein Sci 21: 1298-314 (2012); Madhuranta Ckam et al., Protein Sci 21: 1015-28 (2012); Varadams ettyG et al., J Mol Biol 424: 68-87 (2012); Reichen C et al., J Struct Biol 185: 147-62 (2014)). 404. 404. 404. id="p-404" id="p-404" id="p-404" id="p-404" id="p-404" id="p-404" id="p-404" id="p-404" id="p-404" id="p-404"
[404] For example, numerous alternati scafvefolds have been identif iedwhich bind to the extracel lularreceptor HER2 (see e.g. Wikma nM et al., Protein Eng Des Sei 17: 455-62 (2004); Orlov Aa etal. Cancer Res 66: 4339-8 (2006); Ahlgren S eta l., Bioconjug Chem 19: 235-43 (2008); Feldwis chJ et al., J Mol Biol 398: 232-47 (2010); U.S. patent 5,578,482;s 5,856,110; 5,869,445; 5,985,553; 6,333,169; 6,987,088; 7,019,017; 7,282,365; 7,306,801; 7,435,797; 7,446,185; 7,449,480; 7,560,111; 7,674,460; 7,815,906; 7,879,325; 7,884,194; 7,993,650; 8,241,630; 8,349,585; 8,389,227; 8,501,909; 8,512,967; 8,652,474; and U.S. patent applicat ion 2011/0059090). In additi onto alternati antivebody formats antibody-li, bindingke abilities may be conferre by dnon-proteina ceouscompounds, such, as, e.g., oligome RNArs, molecule DNAs, molecule carbohs, ydrate and s, glycocalyxcalixarenes (see e.g. Sansone F, Casna A,ti Chem Soc Rev 42: 4623-39 (2013)) or partially proteinac eouscompounds, such as, e.g., phenol-formaldehyde cycl oligomeric coupleds with peptides and calixarene-pepti compode sitions (see e.g. U.S. 5,770,380). 405. 405. 405. id="p-405" id="p-405" id="p-405" id="p-405" id="p-405" id="p-405" id="p-405" id="p-405" id="p-405" id="p-405"
[405] Any of the above binding regio structuren mays be used as a component of a molecule of the prese ntinvention as long as the binding region componen hast a dissociation constant of 105־ to 1012־ mole pers liter, preferab lesly thans 200 nanomolar (nM), towar ands extracel targlular biomolecule.et 406. 406. 406. id="p-406" id="p-406" id="p-406" id="p-406" id="p-406" id="p-406" id="p-406" id="p-406" id="p-406" id="p-406"
[406] In certa embodimein nts, the cell-targetin moleculesg of the prese ntinvention comprise a Shiga toxin effector polypeptide of the present invention linked and/or fused to a binding region capable of specifica bindinglly an extracellular part of a targe biomolecult or ean extracellular targe biomolect ule. Extracellula target r biomolecule mays be selected based on numerous criteria, such as a criterion describe hereid n.WO 2016/196344 PCT/US2016/034778 136 B. Extracellul Targetar Biomolecul Boundes by the Binding Regions 407. 407. 407. id="p-407" id="p-407" id="p-407" id="p-407" id="p-407" id="p-407" id="p-407" id="p-407" id="p-407" id="p-407"
[407] In certa embodimein nts, the binding region of a cell-target molecing ules of the prese ntinvention compris aes proteinaceous region capable of binding specifica tolly an extracellular part of a targe biomolecult or ean extracellular target biomolecule, prefera whicbly his physically coupled to the surfa ceof a cell type of intere suchst, as, e.g., a cance cellr tumor, cel l,plasm cela l,infecte ceild or, host cel l harbori anng intracel lulapathogen.r Targe biomolet cules bound by the binding regio ofn a cell-target moleculeing of the prese ntinvention may include biomarke rs over-proportio ornately exclusively present on cance celr ls, immune cell and/s, or cells infected wit hintracellular pathogens, such as, e.g., viruses, bacter fungi,ia, prions, or protozoans. 408. 408. 408. id="p-408" id="p-408" id="p-408" id="p-408" id="p-408" id="p-408" id="p-408" id="p-408" id="p-408" id="p-408"
[408] The ter m"targe biomolt ecule" refers to a biological molecul commoe, nly a proteinaceous molecule or a protein modified by post-transla modifitionalcati ons, such as glycosyla thattion, is bound by a binding region of a cell-targeting molecule of the prese ntinvention resulting in the targeting of the cell-targeti molengcule to a specif iccel l,cell-type, and/or locat ionwithin a multicellul organism.ar 409. 409. 409. id="p-409" id="p-409" id="p-409" id="p-409" id="p-409" id="p-409" id="p-409" id="p-409" id="p-409" id="p-409"
[409] For purposes of the prese ntinvention, the ter m"extracel"lular with regard to a target biomolecul referse to a biomolecul thate has at least a. portion of its structure expose tod the extracellular environment The. exposure to the extracellu lar environmen of ort accessibility to a part of targ biomoleculet couplede to a cell may be empirical determly ined by the skilled worker using methods well known in the art. Non-limitin examg ples of extracellular targe biomolt ecules include cell membrane components transm, embrane spanning protein cells, membrane-anchored biomolecule cell-surs, face-b biomoleculeound ands, secrete biomolecules.d 410. 410. 410. id="p-410" id="p-410" id="p-410" id="p-410" id="p-410" id="p-410" id="p-410" id="p-410" id="p-410" id="p-410"
[410] With regard to the prese ntinvention, the phras "ephysicall coupledy "' when used to describe a target biomolecul meanse covalent and/or non-covalent intermolecula interracti coupleons the targ biomolecet ule, or a portion thereof, to the outside of a cel l,such as a plurality of non-covalent interactions between the targe t biomolecul ande the cell where the energy of eac hsingle interact ision on the order of at least about 1-5 kiloCalorie (e.g.,s electrostatic bonds hydrogen, bonds ionic, bonds Van, der Wall inters act ions,hydrophobic forces, etc.). All integr membraneal proteins can be found physically coupled to a cell membrane as ,wel asl periphera l membrane protei ns.For example, an extracellular targ biomoleculet mighte comprise a transmembra spannine ng region, a lipid anchor, a glycoli anchorpid ,WO 2016/196344 PCT/US2016/034778 137 and/or be non-covale assntlyocia ted(e.g .via non-specifi hydrophobicc interactions and/o lipidr bindin interg acti ons)wit ha factor comprisin anyg one of the foregoing. 411. 411. 411. id="p-411" id="p-411" id="p-411" id="p-411" id="p-411" id="p-411" id="p-411" id="p-411" id="p-411" id="p-411"
[411] Extracell partular ofs targ biomoleculet mayes include various epitopes , including unmodifie polypeptided polypeptidess, modified by the addition of biochemical functional groups and, glycolipids (see e.g. US 5,091,178: EP2431743). 412. 412. 412. id="p-412" id="p-412" id="p-412" id="p-412" id="p-412" id="p-412" id="p-412" id="p-412" id="p-412" id="p-412"
[412] The binding regions of the cell-target moleculesing of the prese ntinvention may be designed or select baseded on numerous criteria, such as the cell-type specif icexpression of thei targetr biomolecul thees, physical localiza tionof thei r target biomolecules with regard to specif iccell types, and/o ther properti ofes their target biomolecule Fors. example, certa ceinll-targeti molecng ules of the prese nt invention compris bindine regionsg capabl ofe bindin gcell-surfa targetce biomolecule thats are expressed at a cellular surface exclusive byly only one cell- type of a species or only one cell-type withi an multicellula organir sm. It is desirable, but not necessary', that an extracel targelular biomolecult be eintrinsical ly' internalized or be readily forc edto interna lizupone interact witing ha cell-targeting molecule of the prese ntinvention. 413. 413. 413. id="p-413" id="p-413" id="p-413" id="p-413" id="p-413" id="p-413" id="p-413" id="p-413" id="p-413" id="p-413"
[413] Among certain embodiments of the cell-targeti molecng ules of the present invention, the bindin regg ion is derived from an immunoglobulin-type polypept ide selected for specif icand high-affinity bindin tog a surfac antigene on the cell surfa ce of a cance orr tumor cel l,where the antigen is restricted in express ionto cance orr tumor cells (see Glokl Jer et al., Molecules 15: 2478-90 (2010): Liu ¥ et al., Lab Chip 9: 1033-6 (2009). In accordance wit hothe embodr iment thes, bindin gregio isn selected for specif icand high-affinity bindin tog a surfac antigene on the cell surfa ce of a cance celr l,where the antigen is over-expre ssedor preferentially express byed cance cellsr as compared to non-cancer cell s.Some represent ativetarge t biomolecule includes but, are not limite to,d the followi enumerang tedtargets associa tedwith cancer and/os specifr icimmune cell types. 414. 414. 414. id="p-414" id="p-414" id="p-414" id="p-414" id="p-414" id="p-414" id="p-414" id="p-414" id="p-414" id="p-414"
[414] Man yimmunoglobulin-type bindin gregions that bind wit hhigh affinity' to extracellular epitopes associated with cance ceilsr are known to the skilled worker, such as binding regions that bind any one of the followi targeng biomolet cule s: annex AI,in B3 melano maantigen, B4 melanoma antigen, CD2, CD3, CD4, CD19, CD20 (B-lymphoc yteantigen protein CD20), CD22, CD25 (interleukin-2 recept or IL2R), CD30 (TNFRSF8), CD37, CD38 (cvcl ADPic ribose hydrolas CD40,e), CD44 (hyaluronan receptor), ITGAV (CD51), CD56, CD66, CD70, CD71 WO 2016/196344 PCT/US2016/034778 138 (transferrin receptor CD73,), CD74 (HLA-DR antigens-assoc invariatediant chain), CD79, CD98, endoglin (END, CD 105), CD 106 (VCAM-1), CD 138, chemokine receptor type 4 (CDCR-4, fusin, CD 184), CD200, insulin-li growthke factor 1 receptor (CD221), mucin l(MUC1, CD227, CA6, CanAg), basal cel adhel sion molecule (B-CAM, CD239), CD248 (endosiaii TEM1)n, , tumor necrosi factors receptor 10b (TNFRSF10B, CD262), tumor necrosi factors receptor 13B (TNFRSF13B, 'TACI, CD276), vascular endothelial growth factor receptor 2 (KDR, CD309), epithelial cell adhesion molecule (EpCAM, CD326), human epidermal growth factor receptor 2 (HER2, Neu, ErbB2, CD340), cance antigenr 15-3 (CA15- 3), cance antigenr 19-9 (CA 19-9), cance antigenr 125 (CA125, MUC16), CA242, carcinoembryonic antigen-relat cell adheed sion molecule (e.g.s CEA CAMS (CD66d) and CEACAM5), carcinoembryonic antigen protein (CEA), choline transporter protein-like 4 (SLC44A4), chondroitin sulfate proteoglyc 4 (CSP4,an MCSP, NG2), CTLA4, delta-like proteins (e.g. DLLS, DLL4), ectonucleotide pyrophosphatase/phosphodieste proteins rase(e.g. ENPP3), endothel receptorsin (ETBRs), epiderma growthl factor receptor (EGFR, ErbBl), folate receptor s (FOLRs, e.g. FRa), G-28, ganglioside GD2, gangliosi GD3,de HLA-DrlO, HLA- DRB, human epiderma growthl factor receptor 1 (HERl) ,HER3/ErbB-3, Ephrin type-B receptor 2 (EphB2), epithel cellial adhesion molecule (EpCAM), fibroblas t activati proteinon (FAP/seprase guanylyl), cyclas c (GCCe ), insulin-li growthke factor 1 recept (IGFor IR), interleukin 2 receptor (IL-2R), interleukin 6 receptor (IL- 6R), integrins alpha-V beta- 3(avP3), integrins alpha-V beta-5 (av|35), integrin s alpha-5 beta- 1(a5P1), L6, zinc transport (LIVer-1), MPG, melanoma-associ ated antigen 1 protein (MAGE-1), melanoma-associa antigented 3 (MAGE-3), mesothel (MSLN)in , metalloreducta.s STEAP1,e MPG, MS4A, NaPi2b, necti ns(e.g. nectin-4), p21, p97, polio virus receptor -like4 (PVRL4), protease-activate d- recepto (sursch as PARI), prostate-spe membrcific ane antigen protei ns(PSMAs), SLIT and N fRK-hke proteins (e.g. SM I RK6). Thomas-Friedenre antigeich n, transmemb raneglycoprotein (GPNMB), trophoblas glycoproteit (TPGB,ns 5T4, WAIF1), and tumor-assoc iatedcalcium signal transducers (TACSTDs, e.g. Trop-2 , EGP-1, etc.) (see e.g. Lui B et al., Cancer Res 64: 704-10 (2004): Novellino L et al., Cancer Immunol Immunother 54: 187-207 (2005); Bagley Ret al., IntJ Oncol 34: 619-27 (2009); Gerbe Hr et al., mAbs 1: 247-53 (2009); Beck A et al., Nat Rev Immunol 10: 345-52 (2010); Anderse J net al., J Biol Chem 287: 22927-37 (2012);WO 2016/196344 PCT/US2016/034778 139 Nolan-Stevaux O et al., PLoS One 7: 650920 (2012); Rust S et al., Moi Cancer 12: 11 (2013)). This list of targe biomolect ules is intend toed be non-limitin Itg. will be appreciat byed the skilled worker that any desired targe biomolect uleassocia tedwith a cance cellr or othe desr ired cell type may be used to design or select a binding region whic hmay be suitable for use as a component of a cell-target moleculeing of the prese ntinvention. 415. 415. 415. id="p-415" id="p-415" id="p-415" id="p-415" id="p-415" id="p-415" id="p-415" id="p-415" id="p-415" id="p-415"
[415] Examples of othe targer biomolecult whiches are strongly associa tedwith cance ceilsr and are bound with high-affinity by a known immunoglobulin- type bindin gregion include BAGE proteins (B melanoma antigens), basal cell adhesion molecules (BCAMs or Luther anblood group glycoprote bladderins), tumor antigen (BTA), cancer-te antigenstis NY-ESO-1, cancer-te antigenstis LAGE proteins, CD19 (B-lymphoc yteantigen protein CD19), CD21 (complement receptor-2 or complement 3d receptor CD26), (dipeptidyl peptidase- DPP4,4, or adenosine deaminase complexing protein 2), CD33 (sialic acid-binding immunoglobulin-type lectin- 3),CD52 (CAMPATH-1 antigen), CD56, CS1 (SLAM famil numbery 7 or SLAMF7), cell surface A33 antigen protein (gpA33), Epstein-Bar virusr antigen protei ns,GAGE/PAGE proteins (melanoma associated cancer/testis antigens), hepatocyte growth factor receptor (HGFR or c-Met), MAGE protei ns,melanoma, antigen recogniz byed T-cel ls1 protein (MART-l/MelanA, MARTI), mucins, Preferentially Expressed Antigen of Melanoma (PRAME) protei ns,prostate specifi c antigen protei (PSA),n prostate stem cell antigen protei (PSCAn ), Receptor for Advance Glycad tion Endroducts (RAGE), tumor-associa glycoprted otein 72 (TAG- 72), vascular endothe lialgrowth factor recepto (VEGFRs),rs and Wilms’ tumor antigen. 416. 416. 416. id="p-416" id="p-416" id="p-416" id="p-416" id="p-416" id="p-416" id="p-416" id="p-416" id="p-416" id="p-416"
[416] Examples of othe targer biomolt ecule whichs are strongly associated with cance cellsr are carbonic anhydrase IX (CA9/CAIX), claudi protein ns(CLDN3, CLDN4), ephrin type- Areceptor 3 (EphA3) ,fola bindingte protei ns(EBP), ganglioside GM2, insulin-like grow thfactor receptor integris, (sunsch as CD1 la-c ), receptor activator of nuclea factorr kappa B (RANK), receptor tyrosine-protei n kinase erB-3, tumor necrosi facs tor receptor 10A (TRAIL-R1/DR4), tumor necros is factor receptor 10B (TRAIL-R2), tenascin C, and CD64 (FcvRI) (,see Hough C et al., Cancer Res 60: 6281-7 (2000); Thepen T et al., Nat Biotechnol 18: 48-51 (2000); Pastan I et al., Nat Rev Cancer 6; 559-65 (2006); Pastan, Annu Rev Med 58: 221-37 (2007); Fitzgera Dld et al., Cancer Res 71: 6300-9 (2011); Scott A et al., CancerWO 2016/196344 PCT/US2016/034778 140 Immun 12: 14-22 (2012)). This list of target biomolecules is intend toed be non- limiting. 417. 417. 417. id="p-417" id="p-417" id="p-417" id="p-417" id="p-417" id="p-417" id="p-417" id="p-417" id="p-417" id="p-417"
[417] In addition, there are numerous othe examplesr of contemplate targetd, biomolecule suchs, as, e.g., ADAM metalloprote (e.g.inases ADAM-9, ADAM-10, ADAM-12, ADAM-15, ADAM-17), ADP-nbosyltransfera (ART1,ses ART4), antigen F4/80, bone marrow strom antia gens (BST1, BST2), break point cluste r region-c-abl oncogene (BCR-ABL) protein C3aRs, (complem entcomponen 3at receptors), CD7, CD 13, CD 14, CD 15 (Lewi sX or stage-speci embrfic yonic antigen 1), CD23 (FC epsilon R1I), CD45 (prote tyrosin inephosphata recese ptor type C), CD49d, CD53, CD54 (intercellula adhesionr molecule 1), CD63 (tetraspa nin), CD69, CD80, CD86, CD88 (complem entcomponen 5at receptor 1), CD115 (colony stimulat facing tor 1 receptor IL-1R), (interleu1kin- receptor) CD123, (interleuk in-3 receptor CD), 129 (interleukin 9 receptor CD), 183 (chemokine recept CXCR3),or CD191 (CCR1), CD193 (CCR3), CD195 (chemokine receptor CCR5), CD203c, CD225 (interferon-induce transmemd brane protein 1), CD244 (Natural Killer Cell Receptor 2B4), CD282 (Toll-like receptor 2), CD284 (Toll-like receptor 4), CD294 (GPR44), CD305 (leukocyte-associa immunoglobulin-ted receplike tor 1), ephrin type-A receptor 2 (EphA2), FceRIa, galectin-9, alpha-fetoprote antigenin 17-Al protein, huma naspart (asparyl aginyl) beta-hydroxyl (HAAH)ase , immunoglobuli n- like transcript ILT-3, lysophosphatidlglycer acyltranol sfe 1 rase (LPGAT1/IAA0205), lysosome-associ membratedane proteins (LAMPs, such as CD107), melanocyte protein PMEL (gplOO), myeloid-relat proteed in-14 (mrp-14), NKG2D ligands (e.g., MICA, MICE, ULBP1, ULBP2, UL-16-binding protei ns,H- 60s, Rae-ls, and homologs thereof), recept tyrosine-or protein kinase erbB-3, SART protei ns,scavenge recr epto (sursch as CD64 and CD68), Siglecs (sialic acid-binding immunoglobulin- lectitype ns), syndecans (such as SDC1 or CD 138), tyrosinas e, tyrosinease-r proteinelated 1 (TRP-1), tyrosinease-relate protein 2d (TRP-2), tyrosinase associate antigend (TAA), APO-3, BCMA, CD2, CD3, CD4, CDS, CD18, CD27, CD28, CD29, CD41, CD49, CD90, CD95 (Fas), CD103, CD104, CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), chemokine receptors, complement proteins cytokine, receptors histoc, ompati bilityprotei ns,ICOS, interferon-al interpha,feron-beta, c-myc, osteoprotegeri PD-1,n, RANK, TACI, INF receptor superfamily member (TNF-R1, TNFR-2), Ap02/TRAIL-Rl ,TRAIL-R2, TRA1L-R3, and TRA1L-R4 (see Scott A et al., Cancer Immunity 12: 14 (2012):WO 2016/196344 PCT/US2016/034778 141 Cheever etal.,M Clin Cancer Res 15; 5323-37 (2009)), for target biomolecules and note the target biomolecules described there arein non-limit ingexamples). 418. 418. 418. id="p-418" id="p-418" id="p-418" id="p-418" id="p-418" id="p-418" id="p-418" id="p-418" id="p-418" id="p-418"
[418] In certa embodimein nts, the binding region compris ores consists essentiall y of an immunoglobulin- bindingtype region capable of specifica bindinglly with high-affinity to the cellular surface of a cell type of the immune system. For example, immunoglobulin-type bindin domainsg are known which bind to immune cell surfac face tors such, as, e.g., CD1, CD2, CD3, CD4, CD5, CD6, CD7, CDS, CD9, CD10, CD11, CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD33, CD34, CD35, CD36, CD37, CD38, CD40, CD41, CD56, CD61, CD62, CD66, CD95, CD117, CD123, CD235, CD146, CD326, interleukin-1 receptor (IL- 1R), interleukin-2 receptor (IL-2R), receptor activator of nuclea factorr kappa B (RANKL), SLAM-associated protein (SAP), and TNFSF18 (tumor necrosi factors ligand 18 orGITRL). 419. 419. 419. id="p-419" id="p-419" id="p-419" id="p-419" id="p-419" id="p-419" id="p-419" id="p-419" id="p-419" id="p-419"
[419] For furthe examplesr of targe biomolt ecules and binding regions envisioned for use in the molecule ofs the present invention, see WO 2005/092917, WrO 2007/033497, US2009/0156417, JP4339511, EP1727827, DE602004027168, EP1945660, JP4934761, EP2228383, US2013/0196928, WO 2014/164680, WO 2014/164693, WO 2015/138435, WO 2015/138452, WO 2015/113005, WO 2015/113007, WO 2015/191764, US20150259428, 62/168,758, 62/168,759, 62/168,760, 62/168,761, 62/168,762, 62/168,763, and PCT/US2016/016580. 420. 420. 420. id="p-420" id="p-420" id="p-420" id="p-420" id="p-420" id="p-420" id="p-420" id="p-420" id="p-420" id="p-420"
[420] It will be appreciated by the skilled worker that any desired target biomolecul maye be used to design or selec a tsuitable bindin gregion to be associated and/or coupled with a Shiga toxin effector polypeptide to produc ae cell - targeting molecule of the present invention. 421. 421. 421. id="p-421" id="p-421" id="p-421" id="p-421" id="p-421" id="p-421" id="p-421" id="p-421" id="p-421" id="p-421"
[421] Any of the above binding regions described herei mayn be used alone or in combinat ionwith each individua embodl iment of the prese ntinvention, including methods of the prese ntinvention. 422. 422. 422. id="p-422" id="p-422" id="p-422" id="p-422" id="p-422" id="p-422" id="p-422" id="p-422" id="p-422" id="p-422"
[422] The general struct ureof the cell-target molecing ules of the prese ntinvention is modular, in that various diverse, ce, ll-targeti bindingng regions may be associated wit hvarious Shiga, toxin effector polypepti desof the prese ntinvention to creat e different, cell-target molecing ules of the present invention which exhibit differences in their cell-targeti actingvitie dues to difference in theirs binding regions This. enables a variet ofy cell-target activitiesing to be exhibited by differ entembodiments WO 2016/196344 PCT/US2016/034778 142 of the cell-targeting molecules of the prese ntinvention such that differ ent embodiments targe difft erent types of cells with Shiga toxin effecto functions,r such as, e.g., cytostas cytotoxicity,is, and intracellula deliver ofry exogenous materia ls.
Furthermore, certa embodimein nts of the cell-targeti molecng ules of the present invention exhibi cert tai charn acter dueist icsto differences in their respective Shiga toxin effector polypeptide regions, such as, e.g., low antigenicit and/ory immunogenicity when administer toed a chordate res, istance to proteol yticcleavage by certain protease highs, stability when administer to eda multicellul organism,ar in vivo tolerability at high dosages, ability to delive a rcargo to an intracellula locatir on, and/o abilr ity to deliver a T-cell epitope to a MHC cla ssI molecule for presentat ion on a cellular surface. 423. 423. 423. id="p-423" id="p-423" id="p-423" id="p-423" id="p-423" id="p-423" id="p-423" id="p-423" id="p-423" id="p-423"
[423] For the purposes of the present invention, the specifi orderc or orientation of the Shiga toxin effecto polypeptider region and the cell-targeti bindingng, region is not fixe din relat ionto eac hother or within the cell-targetin molegcule of the prese nt invention unles expresss noted.ly For example, when the cell-target moleingcule of tire prese ntinvention is a fusion protein wit han amino-termin andal(s) carboxy- terminal(s various), arrangement of thes components of the invention may be suitable (see e.g. Figure 1). In certai embodimn ents of the cell-target moleculesing of the prese ntinvention, the arrangeme of theint compor nents in relat ionto eac h other or within the cell-targeti molengcule are limited as described herein. For example, certain endoplasmic reticulum retention/re trievasigna motifsll are commonly positioned on a carboxy-termi ofnus a. cell-target moleculeing of the present invention and/o ar carboxy-termi ofnus a protein component of a cell- targeting molecule of the present invention.
C. Endoplasm Reticic ulum Retention/Retr Signalieval Motif of a Member of the KDEL Family 424. 424. 424. id="p-424" id="p-424" id="p-424" id="p-424" id="p-424" id="p-424" id="p-424" id="p-424" id="p-424" id="p-424"
[424] Certain embodimen ofts the cell-target molecing ules of the prese ntinvention comprise one or more carboxy-term endoplinal, asm reticulic umretention/ret rieval signa motifl ofs a member of the KDEL Family. Any endoplasmic reticulum retention/re triesignalval motif describe ind WO 2015/138435 may be suitable for use as a component of certai cell-n target moleculesing of the prese ntinvention. [425 ] For purposes of tire prese ntinvention, the phras "eendoplasmic reticulum retention/re triesignalval motif,־’ KDEL-type signa motif,l or signa motifl refers to WO 2016/196344 PCT/US2016/034778 143 any member of the KDEL family capable of functioning within a eukaryoti cecll to promot subcele lular localization of a cell-targeti molecng ule of the prese ntinvention or component thereof to the endoplasmic reticulum via KDEL receptors. 426. 426. 426. id="p-426" id="p-426" id="p-426" id="p-426" id="p-426" id="p-426" id="p-426" id="p-426" id="p-426" id="p-426"
[426] The carboxy-termina lysine-al sparagine-glutam ate-leu(KDEL) sequecine nce (SEQ ID NO:514) is a canonic endoplal asm reticiculum retent ionand retrieval signal motif for soluble proteins in eukaryotic cells and is recogniz byed tire KDEL recepto (serse, Capitani M, Salles M,e FEES Lett 583: 3863-71 (2009), for review).
Hie KDEL family of signal motifs includes many KDEL-Iike motifs, such as HDEL (SEQ ID NO:515), RDEL (SEQ ID NO:516), WDEL (SEQ ID NO:517), YDEL (SEQ ID NO:518), HEEL (SEQ ID X0:5 19). KEEL (SEQ ID NO:520), REEL (SEQ ID NO:521), KEEL (SEQ ID NO:522), KIEL (SEQ ID NO:523), DKEL (SEQ ID NO:524), KKEL (SEQ ID NO:525), HNEL (SEQ ID NO:526), HTEL (SEQ ID NO; 527), KTEL (SEQ ID NO:528), and HVEL (SEQ ID NO:529), all of which are found at the carboxy-termi ofnals proteins which are known to be residen ofts the lumen of the endoplasm reticic ulum of throughout multiple phylogenetic kingdoms (Munr oS, Pelham H, Cell 48: 899-907 (1987); Raykhel I et al., J Cell Biol 179: 1193-204 (2007)). The KDEL signal motif famil includesy at leas 46t polypept ide variant showns using synthet constric ucts (Raykhel, J Cell Biol 179: 1193-204 (2007)). Additional KDEL signa motifsl include ALEDEL (SEQ ID NO:530), HAEDEL (SEQ ID NO:531), HLEDEL (SEQ ID NO:532), KLEDEL (SEQ ID NO:533), IRSDEL (SEQ ID NO:534), ERSTEL (SEQ ID NO:535), and RPSTEL (SEQ ID NO:536) (Alane Hn et al., J Mol Biol 409: 291-7 (2011)). A general ized consensus motif representing the majority of KDEL signa motifl hass been describe d as [KRHQSA]-[DENQ]-E-L (Hulo N et al., Nucleic Acids Res 34: D227-30 (2006)). 427. 427. 427. id="p-427" id="p-427" id="p-427" id="p-427" id="p-427" id="p-427" id="p-427" id="p-427" id="p-427" id="p-427"
[427] Proteins containi KDELng famil signaly motifs are bound by KDEL receptors distributed throughout the Golgi complex and transpo rteto thed endoplasm reticic ulum by a microtubule-depende mechanisnt form release into the lumen of the endoplasm reticic ulum (Griffiths G et al., J Cell Biol 127: 1557-74 (1994); Miesenbock G, Rothma J,n J Cell Biol 129: 309-19 (1995)). KDEL receptors dynamically cycle between tire Golgi complex and endoplasm reticic ulum (Jackson M et al., EMBO J. 9: 3153-62 (1990); Schutze M et al., EMBO J. 13: 1696-1705 (1994)). 428. 428. 428. id="p-428" id="p-428" id="p-428" id="p-428" id="p-428" id="p-428" id="p-428" id="p-428" id="p-428" id="p-428"
[428] For purposes of the prese ntinvention, the members of the KDEL family include synthetic signa motifl ables to function within a eukaryoti cecll to promotesubcellula locar lizati ofon a prote toin the endoplasmic reticulum via KDEL receptors. In other words, some members of the KDEL famil mighty not occur in nature or have yet to be observe ind nature but have or may be construc andted empirical verifiedly by the skilled worker using metho dsknown in the art; see e.g., Raykhel I et al., J Cell Biol 179: 1193-204 (2007). 429. 429. 429. id="p-429" id="p-429" id="p-429" id="p-429" id="p-429" id="p-429" id="p-429" id="p-429" id="p-429" id="p-429"
[429] As a component of certa celin l-target moleingcules of the prese ntinventi on, the KDEL-type signa motl ifis physicall locaty ed,orient ed,or arranged within the cell-targeti molngecule such that it is on a carboxy-termi ofnal a polypeptide component of the cell-targeti molngecul ofe the present invention. 430. 430. 430. id="p-430" id="p-430" id="p-430" id="p-430" id="p-430" id="p-430" id="p-430" id="p-430" id="p-430" id="p-430"
[430] In certai embodimen nts of the cell-targeti moleculng ofes the present invention the, binding region and the Shiga toxi effen ctor polypept ideregion, and/or endoplasm retiic culum retention/ret signrievalal moti mayf be direct lilynked to each other and/or suitably linked to eac hother via one or more intervening component s, such as with one or more linkers well known to the skill edworker and/or described herein.
D. Addition Exogenousal Materials 431. 431. 431. id="p-431" id="p-431" id="p-431" id="p-431" id="p-431" id="p-431" id="p-431" id="p-431" id="p-431" id="p-431"
[431] In certai embn odiments the ,cell-targeti molengcules of the present invention comprises an additional exogenous materi al.An "additional exogenous material" as used herei refersn to one or more atom ors molecules, often not generally present in both Shiga toxins and native target cells, where the cell-target molingecule of the present invention can be used to specifical transly port such material to the interior of a cell In. one sense, the entire cell-target molingecule of the invention is an exogenous materia whichl wil entel ther cel l;thus the, "additional" exogenou s materi alsare heterologous materi alslinked to but other than the core cell-targeti ng molecul itse elf. Non-limiti exampleng ofs additional exogenous materi alsare radionucleide peptidess, detect, ionpromot ingagents, proteins smal, molel cule chemotherapeuti agents,c and polynucleotides. 432. 432. 432. id="p-432" id="p-432" id="p-432" id="p-432" id="p-432" id="p-432" id="p-432" id="p-432" id="p-432" id="p-432"
[432] In certain embodiment of sthe cell-targeti molengcule ofs the present invention, the additional exogenous material is one or more radionucleide suchs, as, e.g., 211At, 131I, 125I, %0y, 111In, 186Re, 188Re, 153Sm, 212Bi, 32P, 60C, and/or radioac tive isotopes of lutetium. 433. 433. 433. id="p-433" id="p-433" id="p-433" id="p-433" id="p-433" id="p-433" id="p-433" id="p-433" id="p-433" id="p-433"
[433] In certain embodiments, the additional exogenous materia comprisl aes proapopto peptide,tic polypepti orde, protein, such as, e.g., BCL-2, caspases (e.g. -144-WO 2016/196344 PCT/US2016/034778 145 fragmen ofts caspase-3 or caspase-6), cytochro mes,granzyme B, apoptosis-induc ing factor (AIF), BAX, tBid (trunca tedBid) ,and proapoptotic fragmen orts derivatives thereof (see e.g., Ellerby H et al., Nat Med 5: 1032-8 (1999); Mai J et al., Cancer Res 61: 7709-12 (2001); Jia L et al., Cancer Res 63: 3257-62 (2003); Liu Y et al., Moi Cancer Ther 2: 1341-50 (2003); Perea S et al., Cancer Res 64: 7127-9 (2004); XuY etaL, J Immunol 173: 61-7(2004); Dalken B et al., Cell Death Differ 13: 576- 85 (2006); Wang T et al., Cancer Res 67: 11830-9 (2007); Kwon M et al., Mol Cancer Ther 1: 1514-22 (2008); Qiu X et al., Mol Cancer Ther 7: 1890-9 (2008); Shan L et al., Cancer Biol Ther 11: 1717-22 (2008); Wang F et al., Clm Cancer Res 16: 2284-94 (2010); Kim J et al., J Virol 85: 1507-16 (2011)). 434. 434. 434. id="p-434" id="p-434" id="p-434" id="p-434" id="p-434" id="p-434" id="p-434" id="p-434" id="p-434" id="p-434"
[434] In certain embodiments, the additional exogenous material compris aes protein or polypeptide comprising an enzyme. In certa othein embodimer nts, the additional exogenou materials is a nuclei acidc such, as, e.g. a ribonucl eicacid that functions as a sma llinhibiting RNA (siRNA) or microRNA (miRNA). In certain embodiments, the additional exogenous material is an antigen, such as antigens derived from pathogens, bacterial protei ns,viral protei ns,proteins mutated in cancer, proteins aberrantly express ined cance orr, T-cell complementary determin regionsing For. example, exogenous materi alsinclude antige ns,such as those characterist of antigeic n-presenting cells infected by bacter andia, T-cell complement detearyrmin regionsing capable of functioning as exogenou antiges ns.
Exogenous materials comprising polypeptides or proteins may optionally comprise one or more antige whetherns known or unknown to the skill edworker. 435. 435. 435. id="p-435" id="p-435" id="p-435" id="p-435" id="p-435" id="p-435" id="p-435" id="p-435" id="p-435" id="p-435"
[435] In certain embodiments of the cell-targeting molecules of the present invention, all heterologous antige and/orns epitopes associated with the Shiga toxin effector polypeptide are arrange in dthe cell-targ etingmolecule amino-terminal to the carboxy-terminus of the Shiga toxin Al fragment regio ofn the Shiga toxin effect or polypeptide. In certa furtherin embodiments, all heterologous antigens and/or epitopes associate withd the Shiga toxin effector polypept ideare associated, either directly or indirectly, with the Shiga toxin effect polypeptideor at a position amino- terminal to the carboxy-terminus of the Shiga toxin Al fragment regio ofn the Shiga toxin effector polypeptide. In certain furthe embodimer nts, all additional exogenou s material(s which) is an antigen is arranged amino-termi tonal the Shiga toxin effect or polypeptide, such as, e.g., fused directly or indirectly to the amino terminus of the Shiga toxin effector polypeptide.WO 2016/196344 PCT/US2016/034778 146 436. 436. 436. id="p-436" id="p-436" id="p-436" id="p-436" id="p-436" id="p-436" id="p-436" id="p-436" id="p-436" id="p-436"
[436] In certa embodimein nts of the cell-targeti moleculeng ofs the present invention, the additional exogenou mates ria is al cytotoxic agent, such as, e.g., a smal molel cule chemotherapeu agent,tic anti-neoplastic agent, cytotoxic antibiotic, alkyla tingagent, antimetabol topoisomite, eras inhibe itor and/or, tubulin inhibitor.
Non-limiting examples of cytotoxic agents suitable for use with the present invention include aziridine cisplatins,s, tetrazines, procarbaz ine, hexamethylmelami vincane, alkaloids, taxanes camptotheci, etoposns, ide, doxorubicin, mitoxantr teniposione, de,novobiocin aci, arubic anthrin, acyc lines, actinomycin, amanitin, amatoxins, bleomycin, centanamyc (indolecin arboxam ide), plicamycin, mitomyci daunorn, ubici epirn, ubici n,idarubicins, dolastatins , maytansines, maytansionoids, duromycin, docetaxel, duocarmycins adri, amyc in, calicheamic aurisin, tati pyrrons, iobenzodiazepine pyrrolobes, nzodiaz dimerepine s (PBDs), carboplat 5-flin,uorourac (5-FU),il capecitabine, mitomycin C, paclitaxel , 1,3-Bis(2-chloroethyl)-l-nitros (BCMourea 1. rifampici cisplatn, methoin, tre xate, gemcitabine aceglat, one,acetogeni (e.g.ns bullatac andin bullatacinone) , aclacinomysi AGns, 1478, AG1571, aldophosphami glycosde ide, alkyl sulfonat es (e.g., busulfa n,improsuifa andn, piposulfan) alkylat, agentsing (e.g. thiotepa and cyclosphospham aminoleide), vulinic acid aminopterin,, amsacrine, ancitabine, anthramycin, arabinosi azade, citidine, azaserine, aziridines (e.g., benzodopa, carboquone, meturedopa, and uredopa) aza, uridi ne,bestrabuc bisantrene,il, bisphosphonates (e.g. clodronate) bleomycins,, bortezomi bryostatib, n, cactinomyci calln, ysta carabitin, cin, carminomycin, carmof ur,carmustine , carzinophilin, CC-1065, chlorambucil chloranbucil, chlomaphaz, chlorozotine, ocin, chromomycinis chromoprot, enediyneein antibioti chromophoresc CPT-1L, cryptophycins (e.g. cryptophyc 1 andin cryptophycin 8), cyclophosphamide, cytarabine, dacarbaz ine,dactinomycin, daunomycin, defofamine, demecolci ne, detorubici diazin, quone, 6-diazo-5-oxo-L-norleuci dideoxyurne, idine, difluoromethylomith (DMFineO), doxifluridine, doxorubicins (e.g., morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinodoxorubi cin, and deoxydoxorubi dynemicicin), ns,edatraxate edatr, exate, eleutherobins , elformithi elline,ptini acetatum enediynee, antibiot (e.g.ics calicheamic ins), enilurac enociil, tabine epir, ubicins epoth, ilone eso, rubicins esp, eramici ns, estramusti ethylene, nimines 2-ethylhydr, azide, etoghicid, fludarabine, folic acid analogues (e.g., denopter methom, tre xatepteropte, andnn, trimetrexate folic ),aci dWO 2016/196344 PCT/US2016/034778 147 replenishe (e.g.rs frolini accid), fotemusti fulvene, str gacytosant, ine,gallium nitrate, gefitinib, gemcitabine hydro, xyure ibandronata, ifose,famide, imatinib mesylate, erlotini fulveb, str letant,rozole PTK787/ZK, 222584 (Novartis, Basel, CH), oxaliplatin, leucovor rapin, amyc lapain, tini lonafamib,b, sorafenib, methylamelamin (e.g.,es altretamine, triethy lenemelamine trie,thy lenephosphorami triethylede, nethiophosphoramide and trimethylomelam ine), pancratista sartins,codictyins spongi, sta tinsnitrog, mustaen rds (e.g., chlorambucil, chlomaphaz cyclophosphamide,ine, mechloretha mechlormine, ethamine oxide hydrochlori melphalde, novembichin,an, phenester prednimusine, tine, trofosfam ide, and urac mustail rd), nitrosurea (e.g.,s carmustine fotemustine,, lomustine, nimustine, and ranimnustine) dynemic, ins, neocarzinostatin chromopho anthres, ramyc in, detorubic epirubin, icins, marcellomycins, mitomycins (e.g. mitomycin C), mycophenolic acid nogalamycins,, olivomyci peplomycins, potfins, romyci ns, puromyci ns,quelamycins, rodorubicins ubenime, x, zinostatins zorabic, ins, purine analo (e.g.,gs fludarabi 6-mene, rcaptopurine thiamiprine, and ,thioguani ne), pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, dideoxyuridi ne, doxifluridine, enocitabine, and floxuridine) ace, glat one,lentinan, lonidainine, maytansinoids (e.g. maytansins and ansamitocins mit),oguazone, mitoxantr one, mopidanmol nitraerine, pentos, tati phenamet,n, pirarubicin, podophyllinic acid 2-, ethylhydrazi rhizoxin,de, sizofuran, spirogermanium, tenuazonic acid tri, aziquone, 2,2',2״trichlorotriethyla trichothecenesmine, (e.g., T-2 toxin, verracur A,in roridin A, and anguidine), urethan, vindesine, mannomustine mitobr, onitol, mitolac tol, pipobroman, arabinosi cycde, lophosphami toxoidsde, (e.g. paclitaxel and doxetaxe l), 6-thioguanine, mercaptopurine, platinum, platinum analogs (e.g. cispla tinand carboplat etoposidein), (VP-16), mitoxantr vinorelone, bine, novantrone, daunomycin, xeloda topoi, someras inhibie tor RFS 2000, retinoids (e.g. retinoic acid), capecitabin lomustine,e, losoxantrone, mercaptopurines, nimustine, nitraerine, rapamycin, razoxane roridin, A, spongistatins streptonigrins,, streptozoc sutentins, , T-2 toxin, thiamiprine, thiotep toxoidsa, (e.g. paclita andxel doxetaxel tuberc), idins, verracurin A, vinblastine, vincristine, and structura analogsl of any of the aforementioned (e.g. synthetic analogs), and/o derir vati ofves any of the aforementioned (see e.g., Lindell T et al., Science 170: 447-9 (1970); Remilla Srd et ah, Science 189: 1002-5 (1975): Ravry M et al., Am J Clin Oncol 8: 148-50 (1985); Ravry M et al., Cancer Treat Rep 69: 1457-8 (1985); Sternberg C et al., Cancer 64:WO 2016/196344 PCT/US2016/034778 148 2448-58 (1989); Bai R et al., Biochem Pharmacol 39: 1941-9 (1990); Boger D, Johnson D, Proc Natl Acad Set USA 92: 3642-9 (1995); Beck J et al., Leuk Lymphoma 41: 117-24 (2001); Cassady J etal., Chern Pharm Bull (Tokyo) 52: 1-26 (2004); Sapr Pa et al., Clin Cancer Res 11: 5257-64 (2005); Okele Ny et al., Cline Cancer Res 16: 888-97 (2010); Oroudjev E et al., Mol Cancer Ther 9; 2700-13 (2010); Ellestad G, Chirality 23: 660-71 (2011); Kantarjian H et al., Lancet Oncol 13: 403-11 (2012); Moldenhauer G et al., J Natl Cancer Inst 104: 622-34 (2012); Meulendijks D et al., Invest New Drugs 34: 119-28 (2016)).
E. Structure-Function Relationships of Cell-Targe Molecting ules of the Invention 437. 437. 437. id="p-437" id="p-437" id="p-437" id="p-437" id="p-437" id="p-437" id="p-437" id="p-437" id="p-437" id="p-437"
[437] For certain embodimen ofts the cell-targetin molecg ules of the prese nt invention, there specif icstructure-function relationships that have been observed, such as, e.g., componen relatt iveorientation effects on cytotoxic potenc ; fiiry in- cleava sensge itivit effey cts on in vivo tolerabili at certaty dosagein furin-s; cleavage sensitivi efftyect ons in vitro stability; furin-cleavage sensitivit effey cts on in vivo half-lif ande; furin-cleava sensitivityge effects on in vivo, non-specif toxicic ityin multicellul organisar ms. 438. 438. 438. id="p-438" id="p-438" id="p-438" id="p-438" id="p-438" id="p-438" id="p-438" id="p-438" id="p-438" id="p-438"
[438] In certain embodiments of the cell-targeti moleculeng ofs the present invention, the specif icorder or orientation of the Shiga toxin effector polypeptide regio andn binding region is fixed such that the bindin gregion is locate withind the cell-targetin molecg ules more proximal to the carboxy-termi ofnus the Shiga toxin effector polypeptide regio thann to the amino-terminus of the Shiga toxin effector polypeptide region. In certai embodimn ents of the cell-targetin moleculesg of the present invention, the arrangeme of thent Shiga toxin effector polypept ideregio n within the cell-targetin molegcule is limited to being at and/or proximal to the amino-terminus of a polypeptide componen oft the cell-targeti molengcule (see Figur e1). For example, certain embodiments of the cell-targeti molengcule of the present invention comprise 1) a binding region oriented within the cell-targetin g molecule at a position carboxy-termina to the lShiga toxin effector polypept ide region, 2) a binding region associated with the Shiga toxin effector polypeptide region at a position dista froml the amino-termin of usthe Shiga toxin effector polypept ideregio (e.g.n distanc ofes 50, 100, 200, or 250 amino acid residues or greate 3)r), a bindin gregion not stericall coveringy the amino-terminus of the Shiga toxin effector polypeptide region, and/or 4) a bindin gregion not sterically hindering WO 2016/196344 PCT/US2016/034778 149 a structur e(snear) the amino-terminus of the Shiga toxin effector polypeptide regio n (see e.g. Figure 1; WO 2015138452). In certain further embodiments, the cell- targeting molecules of the prese ntinvention are capabl ofe exhibiting more optimal cytotoxic potenc suchy, as, e.g., exhibiting a CD50 value which is 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or higher than a relate celld -targetin g refere ncemolecule comprisin theg same Shiga toxin A Subunit effecto polypeptider region(s and) binding region(s wher), ein the binding region is 1) amino-terminal to tiie Shiga toxin A Subunit effector polypeptide region, 2) associa tedwith the Shiga toxin effector polypeptide regio atn a position proximal to the ammo-termi ofnus the Shiga toxin effector polypeptide region (e.g. distanc ofes les thans 50, 40, 30, 20, or amino acid residues or less ),3) not stericall coveringy the amino-terminus of the Shiga toxin effector polypeptide region, and/or 4) not sterically hinderi ang structur e(snear) the ammo-termi ofnus the Shiga toxin effector polypeptide regio n (see e.g. Figure 1; WO 2015/138452). 439. 439. 439. id="p-439" id="p-439" id="p-439" id="p-439" id="p-439" id="p-439" id="p-439" id="p-439" id="p-439" id="p-439"
[439] In certa embodimein nts, the Shiga toxin A Subunit effecto polypeptider of tiie prese ntinvention compris aes Shiga toxin A1 fragment derive regiod n comprising a disrupte furid n-cleava motifge at the carboxy-tenninus of the Shiga toxin A1 fragment derived regio (sucn h as a disrupted furin-cleava sitege locate atd the carboxy-tenni ofnus a Shiga toxin Al fragment region) (see e.g. Figure 1; WO 2015/191764). In certain furthe embodimer nts, the Shiga toxin effecto polypeptr ide is more furin-cleavage resistant as compared to a relate refd ere ncemolecule, such as, e.g., a molecule comprising a wild-type, Shiga toxin A Subunit or Shiga toxin, Al fragment (see e.g. WO 2015/191764). In certain furthe embodimer nts, the Shiga toxin effector polypeptide of tiie present invention exhibits a reduction in furin- cleava reproge ducibly observe tod be 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or less (including 100% for no cleavage) than the furin-cleava of ge a reference molecule observed in the same assa undery the same conditions. In certain further embodiment thes, Shiga toxin effecto polypeptider is more cleavage resistan to at protea othese thanr furin as compared to a relate referenced molecule, such as, e.g., a molecule comprisin a gwild-type, Shiga toxin A Subunit or Shiga toxin Al fragment. 440. 440. 440. id="p-440" id="p-440" id="p-440" id="p-440" id="p-440" id="p-440" id="p-440" id="p-440" id="p-440" id="p-440"
[440] Certai cell-n target moleculeing ofs the prese ntinvention exhibit cytotoxic potencie withins 100-fold, 20-fold 10-fold,, 5-fold, or les thans a refere ncemolecule comprising a wild-type Shiga toxin effector polypeptide region despit thee lack of WO 2016/196344 PCT/US2016/034778 150 any compensator stryuctura fealture for the disrupt edfurin-cleavage motif in die Shiga toxin effector polypeptide. For cell-targetin molecg ules comprisin Shigag toxin A Subunit derived regio nswhic hdo not maintai then forin cleavage event, i.e. molecules comprisin Shigag toxin A Subunit derive componentsd which are not cleaved by furin inside targ ceietls, one alternati forve preservin maximalg cytotoxici is compensation.ty Compensat forion the lack of furin cleavage of a Shiga toxin A Subunit region in cytotoxic molecule might be accomplis hedby presenting the Shiga toxin A Subunit region in a ‘"pre-process" fored m. For example, a cell-target moleculeing comprisi nga Shiga toxin A Subunit region may be construc suchted that the carboxy-terminus of the Shiga toxin A Subunit derived polypeptide is 1) proximal to a carboxy-term ofinus the molecule and 2) matche ors resemble a natives Shiga toxin Al fragment afte clear vage by furi n(see WO 2015/191764). Such compensat ision not require ind certain cell-targeting molecules of the prese ntinvention, rather it is intentiona avoidedlly in order to provide one or more function(s such), as, e.g., improved in vivo tolerabili at cetyrtain dosages increa; sed in vitro stability; increased in vivo half-lif and/oe; redur ced in vivo, non-specifi toxicitc iny multicellular organisms For. certa embodiments,in thes benee ficia functil on(s) are prese ntwithout any significant reduction in cytotoxic potenc ofy the cell-target molecing ule of the present invention as compared to a refere ncemolecule comprising a wild-ty peShiga toxin effector polypeptide. 441. 441. 441. id="p-441" id="p-441" id="p-441" id="p-441" id="p-441" id="p-441" id="p-441" id="p-441" id="p-441" id="p-441"
[441] In certa emboin diments, the cell-target moleingcule of the prese ntinvention compris aes Shiga, toxin A Subunit effector polypeptide comprisi nga Shiga toxin, A1 fragment derived region comprisi nga disrupt edfurin-cieava motifge at the carboxy- termin ofus the Shiga toxin Al fragment derived region (such as a disrupted furin- cleava sitege locate atd the carboxy-terminus of a. Shiga toxin Al fragment region) (see e.g. Figure 1; WO 2015/191764) but do not comprise any compensator y protea clese ava sitege proximal to the carboxy-termi ofnus the Shiga toxin Al fragment derived region and/o orientedr between the Shiga toxin effector polypept ideand a. relatively large mole, cule moiety (e.g. a bindin regiog ofn a size greater than 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, or 50 kDa). In certain furthe embodr iment thes, cell-targetin g molecule of the prese ntinvention compris aes Shiga toxin effector polypept ide which is more furin-cieava resgeist antas compared to a related refere ncemolecule , such as, e.g., a molecule comprisin a gwild-type, Shiga toxin A Subunit or ShigaWO 2016/196344 PCT/US2016/034778 151 toxin Al fragment (see e.g. WO 2015/191764). In certa furthein embodimer nts, the cell-target moleculeing of the prese ntinvention exhibits a reducti inon furin- cleava ofge 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% less than the furin-cleavage of a reference molecule observe ind the same assa y under the same conditions while the cell-target moleingcule exhibits a cytotoxic potency within 100-fold, 20-fold, 10-fold, 5-fold, or les thans a reference molecule comprising a wild-type Shiga, toxin effector polypeptide region. In certain furthe r embodiments, the cell-targetin molegcule of the prese ntinven tion exhibits an improvement in in vivo tolerabili as tycompared to a relate referenced molecule comprising a Shiga, toxin effector polypeptide having a wild-type furin cleava ge motif and/or wild-type furin cleavage site at the carboxy-terminus of its Shiga toxin Al fragment regio (seen e.g. WO 2015/191764). For example, an increas in ein vivo tolerability may be determine by dcomparing measurements of mortality, signs of morbidity, and/o cerr tain clinical signs in groups of laborator animalsy administered different molecules, at the same dosage (sees e.g. Examples, infra; WO 2015/191764). 442. 442. 442. id="p-442" id="p-442" id="p-442" id="p-442" id="p-442" id="p-442" id="p-442" id="p-442" id="p-442" id="p-442"
[442] In certa emin bodiment thes, cell-target moleingcule of the present invention compris aes Shiga toxin A Subunit effector polypeptide comprisi nga Shiga toxin Al fragment derived region comprisi nga disrupt edfurin-cleava motifge at the carboxy- termin ofus the Shiga toxin Al fragment derived region (such as a disrupt edfurin- cleava sitege located at the carboxy-terminus of a Shiga toxin Al fragment derived region) (see e.g. Figure 1; WO 2015/191764). For certain further embodiments, the cell-target moleingcule of the prese ntinvention dial comprise a cytotoxic component, the cell-target moleingcule exhibit reduceds non-specif toxiciic asty compared to more protease-cleava sensgeitive variants which, have great er propens ityto break apar andt there byrelease the cytotoxic component from the bindin gregion, especiall wheny administer to edliving materials such, as, e.g., a population of cells, a tissue, and/or an organism. Furthermo certare, protin eas e- cleava resge ista cellnt, -targeting molecules of the prese ntinventi mayon exhibit, increased, in vivo, half-lives afte adminisr trati to livingon materi als(e.g., certain chordate as s)compared to more protease-cle avagesensitive variant baseds on the protease-cle avageresistance conferr toed the cell-targeti moleculeng by the disrupt edfurin-cleavage motif at the carboxy-termi ofnus the Shiga toxin Al fragment derived region.WO 2016/196344 PCT/US2016/034778 152 III. Linkages Connecting Components of the Invention and/o Theirr Subcomponents 443. 443. 443. id="p-443" id="p-443" id="p-443" id="p-443" id="p-443" id="p-443" id="p-443" id="p-443" id="p-443" id="p-443"
[443] Individual cell-targeti bindinng gregions, Shiga toxin effector polypeptide s, and/o compor nents of the cell-targeti molecng ules present invention may be suitably linked to each othe viar one or more linker wells known in the art and/o descr ribe d herein. Individua polypeptidel subcomponen ofts the bindin gregions, e.g. heavy chain variable regions (Vh), light chain variable regions (VL), COR, and/or ABR regions may, be suitably linked to eac hothe viar one or more linker wels knownl in tiie art and/or describe hereid n.Proteinaceous components of the invention, e.g., multi-chain bindin gregions, may be suitably linked to each othe orr other polypeptide compone ntsof the invention via. one or more linker wells known in the art. Peptide compone ntsof the invention, e.g., KDEL family endoplasm reticic ulum retention/ret signarieval motil fs, may be suitably linked to another component of the invention via one or more linke rs,such as a proteinace linkerous which, are !veil known in the art. 444. 444. 444. id="p-444" id="p-444" id="p-444" id="p-444" id="p-444" id="p-444" id="p-444" id="p-444" id="p-444" id="p-444"
[444] Suitable linke arers generall thosey which allow eac hpolypeptide component of the prese ntinvention to fold with a three-dimensiona struclture ven,׳ similar to the polypept idecomponents produced individual withoutly any linker or other compone nt.Suitable linke includers single amino acids peptide, polypeptidess, and , linker lacs king any of the aforementione suchd, as various non-proteinaceous carbon chains, whether branched or cyclic. 445. 445. 445. id="p-445" id="p-445" id="p-445" id="p-445" id="p-445" id="p-445" id="p-445" id="p-445" id="p-445" id="p-445"
[445] Suitable linke mayrs be proteinaceous and comprise one or more amino acids peptides, and/or, polypeptide Proteinaceouss. linker ares suitable for both recombina fusinton proteins and chemica llylinke conjugad tes A protei. nace ous linker typical hasly from about 2 to about 50 amino acid residues such, as, e.g,, from abou t5 to about 30 or from about 6 to abou t25 amino acid residues The. length of the linker select willed depend upon a variety of factors such, as, e.g., the desired property or properti fores which the linker is being selected . In certain embodiments, the linker is proteinac eousand is linked near the termin ofus a. protein component of the prese ntinvention, typica llywithi aboun t20 amino acids of the terminus. 446. 446. 446. id="p-446" id="p-446" id="p-446" id="p-446" id="p-446" id="p-446" id="p-446" id="p-446" id="p-446" id="p-446"
[446] Suitable linke mayrs be non-proteinac sucheous, as, e.g. chemic allinkers .
Various non-proteinaceous linker knowns in the art may be used to link cell- targeting binding regions to tire Shiga toxin effector polypept idecompone ntsof the WO 2016/196344 PCT/US2016/034778 153 cell-target molecing ules of the present invention, such as linker comms only used to conjugate immunoglobulin polypepti desto heterologous polypeptides. For example, polypeptide regions may be linke usingd the functiona sidel chains of their amino acid residues and carbohydr moietieate suchs as, e.g., a. carboxy, amine sulfhydryl,, carboxyli acidc car, bonyl, hydroxyl, and/o cycr lic ring group. For example, disulfid bondse and thioether bonds may be used to link two or more polypeptides.
In addition, non-natural amino aci dresidues may be used with other functional side chains, such as keton groups.e Example ofs non-proteinac chemieous cal linker s include but are not limited to N-succinimidyl (4-iodoacetyl)-aminobenz >S-(Aoate- , succinimidyl) thioacetate (SATA), N-succinimidyl-oxycarbonyl-cu-methy l-a-(2- pyridyldithio) toluene (SMPT), N-succinimidyl 4-(2-pyridyldithio)-pentanoate (SPP), succinimidyl 4-(N-maleimidomethyl) cyclohexane carboxylate (SMCC or MCC), sulfosuccinimi (4-idylodoacetyl)-ammobenz 4-succinoate, imidyl- oxycarbonyl-a-(2-pyridyldith toluene, sulfio) osuccinimidyl-6-(a-methyl -a- (pyridyldithiol)-toluam hexanoate,ido) N-succinimidyl-3-(-2-pyridyldithio)- proprionate (SPDP), succinimidyl 6(3(-(-2-pyridyldithio)-proprionami do) hexanoate sulfosucc, inimi 6(3(-(-2-pyridyldyl dithio)-propionami hexanoate,do) maleimidocapro (MC),yl maleimidocaproyl-valine-citr ulline-p- aminobenzyloxy carbonyl (MC-vc-PAB), 3-maleimidobenzoic acid TV- hydroxysuccinim esteride (MBS), alpha-alkyl derivatives sulfoN, HS-ATMBA (sulfosuccinimid N-[3-(ylacetylthio)-3-methylbutyryl-beta- alanine]), sulfodichlorophe 2-iminol,nothiolane 3-(2-pyr, idyldithio)-propi hydrazide,onyl Elfman’s reagent, dichlorotriaz acidinic and, S-(2-thiopyridyl)-L-cysteine. 447. 447. 447. id="p-447" id="p-447" id="p-447" id="p-447" id="p-447" id="p-447" id="p-447" id="p-447" id="p-447" id="p-447"
[447] Suitable linkers whether, proteinaceous or non-proteinac mayeous, include , e.g., proteas sensie tive, environme ntalredox potential sensitive pH, sensitive aci, d cleavable, photocleavable, and/or heat sensitive linkers. 448. 448. 448. id="p-448" id="p-448" id="p-448" id="p-448" id="p-448" id="p-448" id="p-448" id="p-448" id="p-448" id="p-448"
[448] Proteinaceous linker mays be chosen for incorporati intoon recombinant fusion cell-targetin molecg ules of the present invention. For recombina fusionnt cell-targetin proteinsg of the invention, linke rstypica llycomprise abou t2 to 50 amino acid residues, preferab aboutly 5 to 30 amino acid residues Commonly. , proteinaceous linke rscomprise a majority of ammo acid residues with polar, uncharged, and/o chargedr residues such, as, e.g., threonine proline,, glutamine, glycine, and alanine. Non-limitin examg ples of proteinace linkeous rsinclude alanine-serme-glycine-glycine-proli (ASGGPne-glutE) (SEQamat IDe NO:538),valine-methionin (VM)e, alanine-methionine (AM), AM(G2to4S)xAM (SEQ ID NO: 539) where G is glycine, S is serine, and x is an integer from 1 to 10. 449. 449. 449. id="p-449" id="p-449" id="p-449" id="p-449" id="p-449" id="p-449" id="p-449" id="p-449" id="p-449" id="p-449"
[449] Proteinaceous linkers may be select baseded upon the propertie desirs ed.
Proteinaceous linkers may be chosen by the skill edworker wit hspecific features in ' mind, such as to optimiz onee or more of the fusion molecul’s foldie ng, stabilit y, expression, solubili ty,pharmacokineti properties,c pharmacodyn propertiamic and/ores, the activit ofy the fused domai nsin the context of a fusion construc as compart toed the activity of the same doma inby itself. For exampl protee, inac eouslinkers may be selecte basedd on flexibility, rigidit and/ory, cleavabili Thety. skilled worker may use databas andes linke designr softwa toolsre when choosing linkers In. certa in embodiments of the present inventi on,linkers may be chosen to optimize expression. In certai embodin ment linkerss, may be chosen to promot intere molecular interactions between identica polypeptidesl or protei tons form homomultimers or different . polypeptides or proteins to form heteromulti mers.For exampl proteie, naceous linkers may be selected which allow for desired non-covalent interactions between polypept ide component of thes cell-targ etimoleculng ofes the invention, such as, e.g., interact ions related to the format iondimers and othe higherr order multimers. 450. 450. 450. id="p-450" id="p-450" id="p-450" id="p-450" id="p-450" id="p-450" id="p-450" id="p-450" id="p-450" id="p-450"
[450] Flexible proteinaceous linkers are often greate thanr 12 amino aci dresidues long and rich in smal non-polarl, amino aci dresidues pola, aminor acid residues, and/or hydrophili amicno acid residues, such as, e.g., glycines serine, ands, threonines.
Flexible proteinac eouslinkers may be chosen to increas thee spati alseparation between component and/ors to allo forw intramolecula interactr ionsbetween components For. exampl varie, ous "GS" linkers are known to the skilled worker and are composed of multipl glycinese and/or one or more serine somets, ime ins repeat ingunits, such as, e.g., (GxS)n (SEQ ID NO:540), (SxG)n (SEQ ID NO:541), (GGGGS)n (SEQ ID NO:542), and (G)n (SEQ ID NO:543), in which x is 1 to 6 and n is 1 to 30. Non-limit ingexampl ofes flexibl proteinacee linkersous include GKSSGSGSESKS (SEQ ID NO:544), EGKSSGSGSESKEF (SEQ ID NO:545), GSTSGSGKSSEGKG (SEQ ID NO:546), GSTSGSGKSSEGSGSTKG (SEQ ID NO:547), GSTSGSGKPGSGEGSTKG (SEQ ID NO:548), SRSSG (SEQ ID NO:549), and SGSSC (SEQ ID NO:550). 451. 451. 451. id="p-451" id="p-451" id="p-451" id="p-451" id="p-451" id="p-451" id="p-451" id="p-451" id="p-451" id="p-451"
[451] Rigid proteinaceous linkers are often stiff alpha-hel strucical tures and rich in proline residues and/or one or more strategica placedlly prolin es.Rigid linkers may be chosen to prevent intramolecula interactr ionsbetween linked components. -154-WO 2016/196344 PCT/US2016/034778 155 452. 452. 452. id="p-452" id="p-452" id="p-452" id="p-452" id="p-452" id="p-452" id="p-452" id="p-452" id="p-452" id="p-452"
[452] Suitable linke mayrs be chosen to allow for in vivo separat ofion components suc,h as, e.g., due to cleava and/oge envirr onment-spec instifiabilic ty.
In vivo cleavable proteinac eouslinke rsare capable of unlinking by proteolyt ic process ingand/or reducing environme oftennts at a specif icsite within an organism or inside a certain cell type. In vivo cleavable proteinaceous linker oftens comprise protea sensse itive motifs and/o disulfider bonds forme byd one or more cysteine pairs. In vivo cleava proteble inac eouslinker mays be designed to be sensitive to proteas thates exist only at certain locati onsin an organism, compartme withints an cell and/o, becomer active only under certain physiological or pathological conditions (such as, e.g., involving protease withs abnormally high levels proteas, es overexpres atsed certain diseas sitese and, proteas speces ifica expresslly byed a pathogenic microorganism) For example,. there are proteinac eouslinker knowns in the art whic hare cleaved by proteas presees ntonly intracellular proteasly, preses ent only withi specifn iccell types, and protease preses ntonly under pathological conditions like cance orr inflammat suchion, as, e.g., R-x-x-R motif and AMGRSGGGCAGNRVGSSLSCGGLNLQAM (SEQ ID NO:551). 453. 453. 453. id="p-453" id="p-453" id="p-453" id="p-453" id="p-453" id="p-453" id="p-453" id="p-453" id="p-453" id="p-453"
[453] In certa embodimein nts of the cell-targeti moleculeng ofs the present invention, a linker may be used which compris onees or more protease sensitive sites to provide for cleava byge a protease present within a targe cellt In. certa in embodiments of the cell-target molecing ules of the invention, a linker may be used which is not cleavable to reduce unwante toxicityd after administra totion a vertebr organiate sm. 454. 454. 454. id="p-454" id="p-454" id="p-454" id="p-454" id="p-454" id="p-454" id="p-454" id="p-454" id="p-454" id="p-454"
[454] Suitable linke mayrs include e.g.,, protea sense sitive, environmental redox potenti senal sitive, pH sensitive, aci dcleavable photocleav, and/orable, heat sensitive linkers, whether proteinace orous non-proteinaceous (see e.g., Doronina. S et al., Bioconjug Chern 17; 114-24 (2003); Saito G et ah, Adv Drug Deliv Rev 55: 199-215 (2003); Jeffrey S et al., J Med Chem 48: 1344-58 (2005); Sanderson R et al., Clin Cancer Res 11: 843-52 (2005); Erickson H et al., Cancer Res 66: 4426-33 (2006); Chen X et al., Adv Drug Deliv Rev 65: 1357-69 (2013)). Suitable cleavable linker mays include linker comprs isi ngcleavable groups which are known in the art. 455. 455. 455. id="p-455" id="p-455" id="p-455" id="p-455" id="p-455" id="p-455" id="p-455" id="p-455" id="p-455" id="p-455"
[455] Suitable linke mayrs include pH sensitive linke rs.For exampl certae, in suitable linker mays be chosen fortheir instabil inity lower pH environme tonts provide for dissociation inside a subcellula comparr tment of a targe cellt (see e.g., van Der Velde Vn et al., Blood 91; 3197-204 (2001); Ulbric K,h Subr V, Adv Drug WO 2016/196344 PCT/US2016/034778 156 Deliv Rev 56: 1023-50 (2004)). For example, linke thatrs comprise one or more trityl groups deriva, tized trityl groups bisma, leimideothoxy propan groupse adipic, acid dihydrazid groupse and/or, acid labile transferr groupsin may, provide for release of compone ntsof the cell-targeti moleculeng ofs the invention, e.g. a polypeptide component, in environme withnts specifi pHc range s.In certai linkern s may be chosen which are cleaved in pH range cors respondi to ngphysiological pH difference betws een tissue s,such as, e.g., the pH of tumor tissu ise lower than in healthy tissues. 456. 456. 456. id="p-456" id="p-456" id="p-456" id="p-456" id="p-456" id="p-456" id="p-456" id="p-456" id="p-456" id="p-456"
[456] Photocleavable linker ares linke rsthat are cleaved upon exposure to electromagnetic radiation of certain wavelength ranges such, as light in the visible range. Photocleavable linke rsmay be used to release a component of a cell- targeting molecule of the invention, e.g. a polypeptide component, upon exposur toe light of certain wavelengt Non-lhs. imiti examng ples of photocleavable linker s include a nitrobenzyl group as a photocleavable protect groupive for cysteine , nitrobenzyloxycar chloridbonyl croe ss-linker hydroxys, propylmethac rylamide copolymer, glycine copolymer, fluoresce copolymer,in and methyirhodam ine copolym er.Photocleavable linker mays have particula users in linking components to form cell-targeti molecng ules of the invention designed for treating diseases , disorde andrs, conditions that can be exposed to light using fiber optics. 457. 457. 457. id="p-457" id="p-457" id="p-457" id="p-457" id="p-457" id="p-457" id="p-457" id="p-457" id="p-457" id="p-457"
[457] In certa embodimein nts of the cell-targeti moleculeng ofs the present invention, a cell-targeti bindinng gregio isn linked to a Shiga toxin effector polypeptide of the present invention using any number of means known to the skilled worke includr, ing both covalent and noncovalent linkages. 458. 458. 458. id="p-458" id="p-458" id="p-458" id="p-458" id="p-458" id="p-458" id="p-458" id="p-458" id="p-458" id="p-458"
[458] In certa embodimein nts of the cell-targeti moleculeng ofs the present invention, the molecule compris aes bindin gregion which is a scFv wit ha linker connecting a heavy chain variable (Vh) domain and a light chain variable (Vl) domai n.There are numerous linke knownrs in the art suitable for tins purpos e,such as, e.g., the 15-residue (Gly4Ser )3peptide (SEQ ID NO:552). Suitable scFv linker s which may be used in forming non-coval multivalentent structure includes GGS (SEQ ID NO:553), GGGS (SEQ ID NO:554), GGGGS (SEQ ID NO:555), GGGGSGGG (SEQ ID NO:556), GGSGGGG (SEQ ID NO:557), GSTSGGGSGGGSGGGGSS (SEQ ID NO:558), and GSTSGSGKPGSSEGSTKG (SEQ IDNO:559).WO 2016/196344 PCT/US2016/034778 157 459. 459. 459. id="p-459" id="p-459" id="p-459" id="p-459" id="p-459" id="p-459" id="p-459" id="p-459" id="p-459" id="p-459"
[459] Suitable methods for linkage of the components of the cell-targeting molecules of the prese ntinvention may be by any method presently known in the art for accomplishi such,ng so long as the attachme doesnt not substantiall impedey the bindin gcapabilit ofy the cell-target bindinging region, the cellular internaliza oftion the Shiga toxin effecto polypeptider component, and/o whenr appropriate the desired Shiga toxin effector function( ass) measured by an appropriate assay, including assa ysdescribed herein. 460. 460. 460. id="p-460" id="p-460" id="p-460" id="p-460" id="p-460" id="p-460" id="p-460" id="p-460" id="p-460" id="p-460"
[460] For the purposes of the ceil-target molecing ules of tire present invention, the specif icorder or orientat ision not fixed for tire component thes: Shiga toxin effector polypeptide( thes) binding, region(s), and any optional linker( ins), relation to eac hother or the entire cell-target moleculeing (see e.g. Figure 1) unless specifica noted.lly The components of the cell-targetin molecg ules of the present invention may be arranged in any order provided that the desired activity( ofies) the binding regio andn Shiga toxin effecto polypeptider are not eliminated.
IV. Examples of Structural Variations of the Shiga Toxin Effector Polypeptides and Cell-Target Molecing ules of the Invention 461. 461. 461. id="p-461" id="p-461" id="p-461" id="p-461" id="p-461" id="p-461" id="p-461" id="p-461" id="p-461" id="p-461"
[461] In certain embodiments, a Shiga toxin effecto polypeptider of the present invention may comprise or consist essentiall of ay truncat Shigaed toxin A Subunit.
Truncations of Shiga toxin A Subunits might resul int the deletion of an enti re epitope(s) and/o epitr ope region(s B-cell), epitope CD4+s, T-cell epitopes and/o, r furin-cleava sitege withouts affect ingShiga toxin effecto functions,r such as, e.g., catalytic activity and cytotoxicity. The smalles Shigat toxin A Subunit fragment shown to exhibit full enzymat actic ivity was a polypept idecomposed of residues 1- 239 of SItlA (LaPointe P et al., J Biol Chern 280: 23310-18 (2005)). Tire smalles t Shiga toxin A Subunit fragment shown to exhibi signift icant enzymat actiic vity was a polypeptide composed of residues 75-247 of StxA (Al-Jaufy X et al., Infect Immun 62: 956-60 (1994)). 462. 462. 462. id="p-462" id="p-462" id="p-462" id="p-462" id="p-462" id="p-462" id="p-462" id="p-462" id="p-462" id="p-462"
[462] Although Shiga toxin effector polypeptides of the prese ntinvention may commonly be smaller than the full-lengt Shigah toxin A Subunit, it is prefer redthat tiie Shiga toxin effector polypeptide region of a cell-target moleculeing of the present invention maintain the polypeptide regio fromn amino acid position 77 to 239 (SLT-1A (SEQ ID NO:1) or StxA (SEQ ID NO:2)) or the equivalent in oilier A Subunits of members of the Shiga toxin family (e.g. 77 to 238 of (SEQ ID NO: 3)).WO 2016/196344 PCT/US2016/034778 158 For example, in certa embodimein nts of the molecules of the present invention, the Shiga toxin effector polypepti desof the prese ntinvention derive fromd SLT-1A may comprise or consist essentiall of aminoy acids 75 to 251 ofSEQ IDNO:1, Ito 241 of SEQ ID NO: 1, 1 to 251 of SEQ ID NO: 1, or amino acids 1 to 261 of SEQ ID NO: 1, wherein relat iveto a wild-type Shiga toxin A Subunit at lea stone amino aci d residue is mutated or has been deleted in an endogenous epitope and/o epitr ope region and/o, whereir theren is a disrupted, furin-cleavage motif region at the carboxy-termi ofnus a Shiga toxin Al fragment derived region. Similarly, Shiga toxin effector polypeptide regions derived from StxA may comprise or consi st essentia oflly amino acid 75s to 251 ofSEQ ID NO:2, 1 to 241 of SEQ ID NO:2, 1 to 251 of SEQ ID NO:2, or ammo acids I to 261 of SEQ ID NO:2, wherei reln ati ve to a wild-type Shiga toxin A Subunit at leas onet amino acid residue is mutated or has been deleted m an endogenous epitope and/or epitope region and/o, whereir n there is a disrupted, furin-cleava motifge region at the carboxy-termi ofnus a Shiga toxin Al fragment derived region. Additionally, Shiga toxin effecto polypeptider regio nsderived from SLT-2 may comprise or consist essentiall of aminoy acid 75s to 251 ofSEQ ID NO: 3, 1 to 241 ofSEQ ID NO:3, 1 to 251 ofSEQ ID NO:3, or amino acid s1 to 261 of SEQ ID NO:3, wherei relatn iveto a. w'ild-type Shiga toxin, A Subunit at least one amino acid residue is mutated or has been deleted in an endogenous epitope and/or epitope region, and/or wherei thern ise a disrupted, furin- cleava motifge regio atn the carboxy-termi ofnus a Shiga toxin Al fragment derived region. 463. 463. 463. id="p-463" id="p-463" id="p-463" id="p-463" id="p-463" id="p-463" id="p-463" id="p-463" id="p-463" id="p-463"
[463] The inventi furtheon provider variantss of Shiga toxin effector polypepti des and cell-targeti molecng ules of the prese ntinvention, wdierein the Shiga toxin effector polypeptide differs from a natural occurrily Shigang toxin A Subunit by only or up to 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or more amino acid residues (but by no more than that which retains at lea st85%, 90%, 95%, 99% or more amino aci dsequence identity'). Thus, a molecule of the present invention derived from an A Subunit of a member of the Shiga toxin, family7 may compri se addition deletions,s, truncations, or other alterations from the original sequence as long as at leas 85%,t 90%, 95%, 99% or more amino acid sequence identit isy maintained to a. natural occurringly Shiga toxin A Subunit and where relatin iveto a wild-type Shiga toxin A Subunit at leas onet amino acid residue is mutat edor has been deleted in an endogenous epitope and/o epitoper region, and/or wherein ther isea disrupted, furin-cleava motige regionf at the carboxy-term ofinus a Shiga toxin Al fragme derivednt region. 464. 464. 464. id="p-464" id="p-464" id="p-464" id="p-464" id="p-464" id="p-464" id="p-464" id="p-464" id="p-464" id="p-464"
[464] Accordingl in y,certai embodimn ents, the Shig atoxin effector polypeptide of a molecule of the present invention compris ores consis essentts ial ofly amino acid sequence havings at leas 55%,t 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 99.7% overal sequl ence identity to a natural occuly rrin Shigg a toxin A Subunit, such as SLT-1A (SEQ ID NO:1), StxA (SEQ ID NO:2), and/or SLT-2A (SEQ ID NO:3) where relatin iveto a wild-type Shiga toxin A Subunit at least one amino acid residue is mutated or has been deleted in an endogenous epitope and/or epitope region, and/or where therein is a disrupted, furin-cleava ge moti regionf at the carboxy-terminus of a Shiga toxi Aln fragment derived region. 465. 465. 465. id="p-465" id="p-465" id="p-465" id="p-465" id="p-465" id="p-465" id="p-465" id="p-465" id="p-465" id="p-465"
[465] Optionally, either a full-leng or tha truncat versied on of the Shig atoxi An Subunit may compri sethe Shiga toxin effect polypeptideor region of a molecule of the present invention, where thein Shiga toxin derived polypeptide compris onees or more mutations (e.g. substitutio deletins, ons, insertions or inve, rsions) as compared to a natura occully rrin Shigg atoxin It. is preferred in certain embodiment ofs the invention that the Shiga toxin effector polypeptides have sufficient sequence identit y to a natura occurringlly Shiga toxin A Subuni tto retai cytotn oxici afterty entry into a cell either, by well-known methods of host cell transform atitraon,nsfection, infection or induction, or by internaliza meditionated by a cell-targeti bindingng region linked wit hthe Shiga toxin effector polypeptide. The most critical residues for enzymat activic ityand/or cytotoxici in thety Shiga toxi An Subunits have been mappe dto the follow resingidue-positi ons:asparagine-75, tyrosine-77, glutamat e- 167, arginine-170, and arginine-176 among othe rs(Di R et al., Toxicon 57: 525-39 (2011)). In any one of the embodiments of the inventi on,the Shig atoxin effector polypeptide mays preferabl buty not necessarily maintai onen or more conserved amino acids at positions, such as those found at positions 77, 167, 170, and 176 in StxA, SLT-1 A, or the equival entconserved position in other members of the Shiga toxin family which are typical requirely ford cytotoxic activity. The capacity of a cytotoxi molecc ule of the invention to caus ecell death, e.g. it scytotoxicit mayy, be measured using any one or more of a number of assa yswell known in the art. -159-WO 2016/196344 PCT/US2016/034778 160 A. Examples of De-immunize d,Shiga Toxin Effect Polypeptor ides 466. 466. 466. id="p-466" id="p-466" id="p-466" id="p-466" id="p-466" id="p-466" id="p-466" id="p-466" id="p-466" id="p-466"
[466] In certa embodimein nts, the de-immunized, Shiga toxin effector polypept ide of the prese ntinvention may consist essentia oflly a trunca tedShiga toxin A Subunit having two or more mutations Truncat. ions of Shiga, toxin A Subunits might result in the deletion of an entire epitope(s and/or) epitope region(s), B-cel epitopel s, CD4+ T-cell epitopes and/or, furin-cleava sitesge without affect ingShiga toxin effector functions, such as, e.g., catalytic activity and cytotoxicity'. Truncating the carboxy-terminus of SLT-1A, StxA, or SLT-2A to amino acids 1-251 remove twos predicted B-celi epitope regions, two predicted CD4 positive (CD4+) T-cell epitopes and, a predicted discontinuou B-cells epitope. Truncatin theg amino- terminus of SLT-1 A, StxA, or SLT-2A to 75-293 removes at leas threet predicted B-cell epitope regions and three predicted CD4++ T-cell epitopes Trunc. ating both amino- and carboxy-termi ofnals SLT-1A, StxA, or SLT-2Ato 75-251 deletes at lea stfive predicted B-cell epitope regions, four putati veCD4+ T-cell epitopes and one predicted discontinu B-ceous ll epitope. 467. 467. 467. id="p-467" id="p-467" id="p-467" id="p-467" id="p-467" id="p-467" id="p-467" id="p-467" id="p-467" id="p-467"
[467] In certa embodimein nts, a de-immunized, Shiga toxin effector polypeptide of the prese ntinvention may comprise or consi stessential ofly' a full-length or trunca tedShiga toxin A Subunit with at leas onet mutation (relat iveto a wild-type Shiga toxin polypeptide) e.g. ,deleti on,insertion, inversion, or substitution, in a provided, endogenous B-ce, ll and/or CD4+ T-cell epitope region. In certai n embodiments, the Shiga toxin effector potypeptide of the present invention compris aes disruption which compris aes mutation (relat iveto a wild-type Shiga, toxin poh'peptide whic) hincludes a deletion of at leas onet amino acid residue within the endogenou B-cells, and/or CD4+ T-cell epitope region In. certain embodiments, the Shiga toxin, effector polypeptide of the prese ntinvention compris aes disrupti onwhich compris anes insertion of at leas onet amino aci d residue withi then endogenous, B-cell and/o CD4+r T-cell epitope region In. certain embodiments, the Shiga toxin effector polypeptide of the present invention compris aes disruption which compris anes inversion of amino acid residues , wherein at least one inverted amino acid residue is within the endogenou B-ces, ll and/o CD4+r T-cell epitope region. In certa embodimein nts, the Shiga toxin effector polypeptide of the present invention compris a.es disrupti onwhich compris aes mutation (relat iveto a wild-type Shiga toxin polypeptide) such, as, e.g., an amino aci dsubstitution, an amino aci dsubstitution to a non-standar ammod acid and/or, an WO 2016/196344 PCT/US2016/034778 161 amino acid residue with a chemical modifiely sided chain. Non-limitin examplesg of de-immunized, Shiga toxin effecto sub-rer gions suitable for use in the present invention are described in WO 2015/113005, WO 2015/113007 and WO 2015/191764. Numerous non-lim, iti examng ples of Shiga toxin effector polypepti desof the prese ntinvention which comprise amino acid substitutions are provided in the Examples. 468. 468. 468. id="p-468" id="p-468" id="p-468" id="p-468" id="p-468" id="p-468" id="p-468" id="p-468" id="p-468" id="p-468"
[468] In othe embodimer nts, the de-immunized, Shiga toxin effector polypeptide of tire prese ntinvention compris aes trunca tedShiga toxin A Subunit which is shorter than a full-length Shiga toxin A Subunit wherein at leas onet amino acid residue is disrupte ind a natively positioned, B-cel and/orl CD4+ T-cell epitope regio n provided in the Examples (see e.g. Tables 1-7 and/or Table B). 469. 469. 469. id="p-469" id="p-469" id="p-469" id="p-469" id="p-469" id="p-469" id="p-469" id="p-469" id="p-469" id="p-469"
[469] To create a de-immunize Shigad, toxin effector polypeptide in principle, modifyi ngany amino acid residue in a provid edepitope regio byn various means can result in a disruption of an epitope, such as, e.g., a modification which, represents a deleti on,insertion, inversion, rearrangeme subsnt,titution, and chemica l modification of a side chain relat iveto a wild-type Shiga toxin polypeptide .
However modify, ing certai amin no aci dresidues and using certain amino acid modifications are more likely to successfully reduce antigenicit and/y or immunogenicit whiley maintaining a certain leve ofl a Shiga toxin effector function( s).For example, terminal truncatio andns internal amino acid substituti ons are preferre becad use these types of modificat ionsmainta thein overall spacing of the amino aci dresidues in a. Shiga toxin effector polypeptide and thus are more likely to mainta Shigain toxin effector polypeptide struct ureand function. 470. 470. 470. id="p-470" id="p-470" id="p-470" id="p-470" id="p-470" id="p-470" id="p-470" id="p-470" id="p-470" id="p-470"
[470] Among certain embodiments of the present invention, the de-immunize d, Shiga, toxin effector polypeptide comprising or consisting essentia oflly amino acid s 75 to 251 of SLT-1A (SEQ ID NO: 1), StxA (SEQ ID NO:2), and/o SLT-2Ar (SEQ ID NO:3) wherei atn lea stone amino acid residue is disrupted in a natively positioned, epitope regio providedn in the Examples (see e.g. 'Tables 1-7 and/o 12).r Among certain othe embodimr ents are de-immunize d,Shiga toxin effector polypepti deswhich compris ore consist essentially of amino acid s1 to 241 of SET- 1A (SEQ ID NO: 1), StxA (SEQ ID NO:2), and/o SLT-2Ar (SEQ ID NO:3) wherein at leas onet amino acid residue is disrupted in a native positily oned, epitope regio n provided in the Examples (see e.g. Table 1-7s and/or 12). Further embodiments are de-immunize d,Shiga toxin effector polypeptides which comprise or consi stWO 2016/196344 PCT/US2016/034778 162 essentially of amino acids 1 to 251 of SLT-1A (SEQ ID NO: 1), StxA (SEQ ID NO:2), and/o SLT-2Ar (SEQ ID NO; 3) wherein at leas onet ammo acid residue is disrupted in a natively positioned, epitope region provided in the Example (ssee e.g.
Tables 1-7 and/or 12). Further embodiments are Shiga toxin effector polypeptides comprisin ammog acid s1 to 261 of SLT-1A (SEQ ID NO:1), StxA (SEQ ID NO:2), and/o SLT-2Ar (SEQ ID NO;3) wherein at least one ammo acid residue is disrupted in a native positily oned, epitope region provid edin the Examples (see e.g. Tables 1- 7 and/o 12).r 471. 471. 471. id="p-471" id="p-471" id="p-471" id="p-471" id="p-471" id="p-471" id="p-471" id="p-471" id="p-471" id="p-471"
[471] There are numerou dives, rse, intern aminoal acid substitutions that can be used to create de-immunized, Shiga toxin effector polypepti desof the invention. Of the possible substitute amino acid tos use withi ann epitope region, the follow ing substitute amino acid residues are predicted to be the mos tlikely to reduce the antigenicit and/ory immunogenicit of any epitope — G, D, E, S, T, R, K, and H.
Except for glycine, thes aminoe acid residues may all be classifi ased polar and/or charged residues Of. the possible amino acid tos substitute with, the following amino acids A, G, V, L, I, P, C, M, F, S, D, N, Q, H, and K are predicted to be the most likely to reduce antigenicit and/oy immunor genicit whiley providing the retention of a significant leve ofl a Shiga toxin, effector function(s depending), on the amino acid substituted for. General thely, substitution should change a polar and/or charged amino acid residue to anon-polar and uncharged residue (see e.g. WO 2015/113005). In addition, it may be beneficia to lepitope disruption to reduce the overa sizlle and/or length of the amino acid residue’s R-group functiona sidel chain (see e.g. WO 2015/113005). However despite these generalities of substitutions most likely to confer epitope disruption, because the aim is to preserve significant Shiga, toxin effector function(s the), substitute amino acid might be more likel toy preser veShiga toxin effector function(s) if it resemble thes amino acid substituted for, such as, e.g., a nonpolar and/or uncharge residued of similar size substituted for a polar and/or charged residue. 472. 472. 472. id="p-472" id="p-472" id="p-472" id="p-472" id="p-472" id="p-472" id="p-472" id="p-472" id="p-472" id="p-472"
[472] In the Examples below and in WO 2015/113005, many mutations have been empirica llytested for effect(s on) the Shiga toxin effector function of vario usShiga toxin effector polypepti desand cell-targeting molecules. Table B summarizes the results describe ind the Example ands in WO 2015/113005 wher ane amino aci d substitution, alone or in combinati witon hone or more othe substitutions,r did not prevent the exhibition of a potent leve ofl a Shiga toxin effector function( s).TableWO 2016/196344 PCT/US2016/034778 163 B uses the epitope regio numben ring schem desce ribe ind the Examples belo w(see Example !-Table 7, infra).
TABLE B. Amino Acid Substitutions in Shiga. Toxin Effector Polypeptides natively positioned ammo acid positions Epitope Region Disrupted Substitution B-Cel lEpitope Region T-Cell Epitope 1 KIA 1-15 1 KIM 1-15 1 T4I 1-15 4—33 1 D6R 1-15 4-33 1 S81 4-33 1-15 1 4-33 T9V 1-15 1 T9I 1-15 4-33 1 K11A 1-15 4-33 1 1-15 K11H 4-33 1 T12K 4-33 1-15 2 4-33 S33I 27-37 2 S33C 27-37 4™ 33 39-48 3 S43N 34-78 39-48 3 G44L 34-78 3 T45V 39-48 34-78 3 T45I 39-48 34-78 3 S45V 39-48 34-78 3 S45I 39-48 34-78 3 G46P 39-48 34-78 3 D47G 39-48 34-78 3 D47M 39-48 34-78 3 N48V 39-48 34-78 3 N48F 39-48 34-78 - L49A immunogenic residue 34-78 - F50T 34-78 - 34-78 A51V 4 D53A 53-66 34-78 4 34-78 D53G 53—66 4 D53N 53-66 34-78 4 V54L 53-66 34-78 4 53-66 34-78 V54I 4 53-66 34-78 R55A 4 53-66 R55V 34-78 4 R55L 53—66 34-78 4 53-66 G56P 34-78WO 2016/196344 PCT/US2016/034778 164 4 I57M 53-66 34-78 4 7F 53-66 34-78 4 53-66 D58A 34-78 4 53-66 D58V 34-78 4 D58F 53-66 34-78 4 P59A 53—66 34-78 4 53-66 P59F 34-78 4 53-66 E60I 34-78 4 E60T 53-66 34-78 4 E60R 53-66 34-78 4 E61A 53-66 34-78 4 53-66 E61V 34-78 4 53-66 34-78 E61L 4 G62A 53-66 34-78 R84A 77-103 77-103 V88A 94-115 D94A 77-103 94-115 S96I 77-103 94-115 T104N 94-115 A105L 94-115 T107P 94-115 L108M 94-115 SI 09 V 94-115 G1WA 94-115 D1HT 94-115 SI 12V 6 141-153 128-168 D141A 6 G147A 141-153 128-168 V154A 128-168 / R179A 179-190 160-183 7 179-190 T180G 160-183 n 1 T181I 179-190 160-183 ר DI 83 A 179-190 160-183 ך 179-190 D183G 160-183 ו 179-190 D184A / D184F 179-190 7 179-190 LI 85 V 7 179-190 S186A 7 179-190 S186F 7 G187A 179-190WO 2016/196344 PCT/US2016/034778 165 7 G187T 179-190 7 179-190 R188A 7 179-190 R188L / 179-190 S189A - DI 98 A immunogenic residue R205A immunogenic residue C242S 236-258 8 R248A 243-257 236-258 8 243-257 236-258 R251A 473. 473. 473. id="p-473" id="p-473" id="p-473" id="p-473" id="p-473" id="p-473" id="p-473" id="p-473" id="p-473" id="p-473"
[473] Based on the empirica evidencel in the Examples here inand in WO 2015/113005, certai amin no acid positions in the A Subunit sof Shiga toxins are predicted to tolera epitte ope disruptions while still retaining significant Shiga toxin effector functions. For exampl thee, followi nativeng occurringly position tolerats e amino acid substitutions, either alone or in combination, whil retaininge a Shiga toxin effector function( suchs) as cytotoxici — ty1 of SEQ ID NO: I or SEQ ID NO:2; 4 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 8 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID N():3; 9 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 11 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 33 of SEQ ID NO: 1 or SEQ ID NO:2; 43 of SEQ ID NO: 1 or SEQ ID NO:2; 44 of SEQ ID NO: 1 or SEQ ID NO:2; 45 of SEQ ID NO: 1 or SEQ ID NO:2; 46 of SEQ ID NO: 1 or SEQ ID NO:2: 47 of SEQ ID NO:1 or SEQ ID NO:2: 48 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQ ID NO: 1 or SEQ ID NO:2; 50 of SEQ ID NO: 1 or SEQ ID NO:2; 51 of SEQ ID NO: 1 or SEQ ID NO:2; 53 of SEQ ID NO: 1 or SEQ ID NO:2; 54 of SEQ ID NO: 1 or SEQ ID NO:2; 55 of SEQ ID NO: 1 or SEQ ID NO:2: 56 of SEQ ID NO: 1 or SEQ ID NO:2; 57 of SEQ ID NO: 1 or SEQ ID NO:2; 58 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 59 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 60 of SEQ ID NO: 1 or SEQ ID NO:2; 61 of SEQ ID NO: 1 or SEQ ID NO:2: 62 of SEQ ID NO:1 or SEQ ID NO:2; 84 of SEQ ID NO: I or SEQ ID NO:2; 88 of SEQ ID NO: 1 or SEQ ID NO:2; 94 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 96 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 104 of SEQ ID NO: 1 or SEQ ID NO:2; 105 of SEQ ID NO: 1 or SEQ ID NO:2: 107 of SEQ ID NO: lor SEQ IDNO:2; 108 of SEQ ID NO: lor SEQ ID NO:2: 109 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 110 of SEQ ID NO: 1 or SEQ ID NO:2; 111 of SEQ ID NO: 1 or SEQ ID NO:2; 112 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ IDNO:3; 141 ofSEQIDNO:! orSEQIDNO:2; 147 of SEQ ID NO: 1, SEQWO 2016/196344 PCT/US2016/034778 166 ID NO:2, or SEQ ID NO:3; 154 of SEQ ID NO:1 or SEQ ID NO:2: 179 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3: 180 of SEQ ID NO:1 or SEQ ID NO:2; 181 of SEQ ID NO: 1 or SEQ ID NO:2; 183 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NOG; 184 of SEQ ID NO: i, SEQ ID NOG, or SEQ ID NO:3; 185 of SEQ ID NO: 1 or SEQ ID NOG; 186 of SEQ ID NO: I, SEQ ID NOG, or SEQ ID NO:3; 187 of SEQ IDNO:1 or SEQ ID NOG; 188 of SEQ IDNO:1 orSEQ ID NOG; 189 ofSEQ IDNO:1 or SEQ ID NOG; 198 ofSEQ IDNO:1 orSEQ ID NOG: 204 of SEQ ID NOG; 205 ofSEQ ID NO:1 or SEQ ID NO:2; 241 ofSEQ ID NO:3; 242 ofSEQ ID NO: 1 or SEQ ID NOG; 247 of SEQ ID NO: 1 or SEQ ID NOG; 247 of SEQ ID NO:3; 248 of SEQ ID NO: 1 or SEQ ID NOG; 250 of SEQ ID NO:3; 251 of SEQ ID NO: 1 or SEQ ID NOG; 264 of SEQ ID NO: i, SEQ ID NOG, or SEQ ID NOG; 265 of SEQ ID NO: 1 or SEQ ID NOG; arid 286 of SEQ ID NO: 1 or SEQ ID NOG. 474. 474. 474. id="p-474" id="p-474" id="p-474" id="p-474" id="p-474" id="p-474" id="p-474" id="p-474" id="p-474" id="p-474"
[474] The empirical data in the Example ands in WO 2015/113005 point towards other epitope disrupti ngsubstituti onsand combinations of epitope disrupting substitutions that can reduce antigenicit and/oy immr unogenicit of ay Shiga toxin effector polypeptide while retaini theng ability of the Shiga toxin effector polypeptide to exhibi at significant Shiga toxin effector function such as, e.g., new combinations of the aforementioned truncations and positions tolerati ng substitutions as wel asl new substitutions at identica posil tion ors conserved position ms related Shiga toxin A Subunits. 475. 475. 475. id="p-475" id="p-475" id="p-475" id="p-475" id="p-475" id="p-475" id="p-475" id="p-475" id="p-475" id="p-475"
[475] It is predictable that othe aminor aci dsubstituti onsto amino aci dresidues of a conserva functitive onal group of a substitution tested herei mayn reduce antigeni cityand/o immunor genicit whiley presenting a significant Shiga toxin effector function. For example, other substitutions known to the skilled worker to be similar to any of KIA, KIM, T4I, D6R, SSI, T8V, T9I, S9I, KI 1 A, KI 1H, T12K, S33I, S33C, S43N, G44L, S45V, S45I, T45V, T45I, G46P, D47M, D47G, N48V, N48F, L49A, F50T, A51V, D53A, D53N, D53G, V54L, V54I, R55A, R55V, R55L, G56P, I57F, I57M, D58A, D58V, D58F, P59A, P59F, E60I, E60T, E60R, E61A, E61V, E61L, G62A, R84A, V88A, D94A, S96I, T104N, A105L, T107P, L108M, S109V, T109V, G110A, D111T, S112V, D141A, G147A, V154A, R179A, T180G, T181I, D183A, D183G, D184A, D184A, D184F, L185V, L185D, S186A, S186F, G187A, G187T, R188A, R188L, S189A, D198A, R204A, R205A, CG42S, S247I, Y247A, R248A, R250A, R251A, or D264A, G264A, T286A, and/o T286Ir may disrup ant endogenous epitope while maintaining at leas onet Shiga toxin effector WO 2016/196344 PCT/US2016/034778 167 function. In particular ami,no acid substitutions to conserva amitiveno acid residues simila tor KIA, KLM, T4I, SSL T8V, T9I, S9I, KHA. KHIL S33I, S33C, S43N, G44L, S45V, S45I, T45V, T45I, G46P, D47M, N48V, N48F, L49A, A51V, D53A, D53N, V54L, V54I, R55A, R55V, R55L, G56P, I57F, I57M, D58A, D58V, D58F, P59A, E60I, E60T, E61 A, E61V, E61L, G62A, R84A, V88A, D94A, S96I, T104N, T107P, L108M, S109V, T109V, GHOA. D111T, S112V, D14LA, GI47A, V154A, R179A, T180G, T181I, D183A, D183G, D184A, D184F, L185V, S186A, S186F, G187A, R188A, R188L, S189A, D198A, R204A, R205A, C242S, S247I, Y247A, R248A, R250A, R251A, D264A, G264A, T286A, and T286I may have the same or similar effects. In certai embodimen nts, a Shiga toxin effector polypeptide of the invention may comprise simila conservatr aminoive acid substitutions to empirica lly tested ones, such as, e.g., KI to G, V, L, I, F, and H; T4 to A, G, V, L, F, M, and S; S8 to A, G, V, L, F, and M; T9 to A, G, L, F, M, and S; S9 to A, G, L, I, F, and M; KI 1 to G, V, L, I, F, and M; S33 to A, G, V, L, F, and M; S43 to A, G, V, 1 . I, F, and M; S45 to A, G, L, F, and M; T45 to A, G, L, F, and M; D47 to A, V, L, I, F, S, and Q; N48 to A, G, L, and M; L49 to G; ¥49 to A; D53 to V, L, I, F, S, and Q; R55 to G, I, F, M, Q, S, K, and H; D58 to G, L, I, S, and Q; P59 to G; E60 to A, G, V, L, F, S, Q, N, D, and M; E61 to G, I, F, S, Q, N, D, M, and R; R84 to G, V, L, I, F, M, Q, S, K, and H; V88 to G; 188 to G; D94 to G, V, L, I, F, S, and Q; S96 to A, G, V, L, F, and M; T107 to A, G, V, L, I, F, M, and S; S107 to A, G, V, L, I, F, and M; S109 to A, G, I, L, F, and M; T109 to A, G, I, L, F, M, and S; S112 to A, G, L, I, F, and M; D141 to V, L, I, F, S, and Q; V154 to G; R179 to G, V, L, I, F, M, Q, S, K, and H; T180 to A, V, L, I, F, M, and S; T181 to A, G, V, L, F, M, and S; D183 to V, L, I, F, S, and Q; DI84 to G, V, L, I, S, and Q; SI86 to G, V, I, L, and M; RI88 to G, V, I, F, M, Q, S, K, and H; SI89 to G, V, I, L, F, and M; DI 97 to V, L, I, F, S, and Q; DI98 to A, V, L, I, F, S, and Q; R204 to G, V, L, I, F, M, Q, S, K, and H; R205 to G, V, L, I, F, M, Q, S, K and H; S247 to A, G, V, I, L, F, and M; ¥247 to A, G, V, L, I, F, and M; R248 to G, V, L, I, F, M, Q, S, K, and H; R250 to G, V, L, I, F, M, Q, S, K, and H; R251 to G, V, L, I, F, M, Q, S, K, and H; D264 to A, G, V, L, I, F, S, and Q; and T286 to A, G, V, L, I, F, M, and S. 476. 476. 476. id="p-476" id="p-476" id="p-476" id="p-476" id="p-476" id="p-476" id="p-476" id="p-476" id="p-476" id="p-476"
[476] Similarly, amino acid substitutions which remove charge, polari ty,and/o r reduce side chai lengthn can disrup ant epitope while maintaining at lea stone Shiga toxin effector function. In certain embodiments, a Shiga toxin effecto polypeptr ide of the invention may compris onee or more epitopes disrupt edby substitutions such WO 2016/196344 PCT/US2016/034778 168 that side chain charge is removed, polarity is removed, and/or side chai nlength is reduced such as, e.g., substituting the appropriate amino acid selected from the following group A, G, V, L, I, P, C, M, F, S, D, N, Q, H, or K for the amino aci d residue at position 1 of SEQ ID NO: 1 or SEQ ID NO:2; 4 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO;3; 6 of SEQ ID NO: 1 or SEQ ID NO:2; 8 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3: 9 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3: 11 of SEQ ID NO: 1, SEQ ID XO: 2 or SEQ ID NO:3; 12 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 33 of SEQ ID NO: 1 or SEQ ID NO:2; 43 of SEQ ID NO: I or SEQ ID NO:2: 44 of SEQ ID NO: 1 or SEQ ID NO:2; 45 of SEQ ID NO: 1 or SEQ ID NO:2; 46 of SEQ ID NO: 1 or SEQ ID NO:2; 47 of SEQ ID NO: 1 or SEQ ID NO:2; 48 of SEQ ID NO: I, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQ ID NO: 1 or SEQ ID NO:2; 50 of SEQ ID NO: 1 or SEQ ID NO:2; 51 of SEQ ID NO: 1 or SEQ ID NO:2: 53 of SEQ ID NO: 1 or SEQ ID NO:2: 54 of SEQ ID NO: 1 or SEQ ID NO:2: 55 of SEQ ID NO: 1 or SEQ ID NO:2; 56 of SEQ ID NO: 1 or SEQ ID NO:2; 57 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 58 of SEQ ID NO: I, SEQ ID NO:2, or SEQ ID NO:3; 59 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3: 60 of SEQ ID NO: 1 or SEQ ID NO:2; 61 of SEQ ID NO: 1 or SEQ ID NO:2; 62 of SEQ ID NO:1 or SEQ ID NO:2; 84 of SEQ ID NO:1 or SEQ ID NO:2; 88 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 94 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 96 of SEQ ID NO: I, SEQ ID NO:2, or SEQ ID NO:3: 104 of SEQ ID NO: 1 or SEQ ID NO:2; 105 of SEQ ID NO: 1 or SEQ ID NO:2; 107 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 108 of SEQ ID NO: 1 or SEQ ID NO:2; 109 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 110 of SEQ ID NO:1 or SEQ ID NO:2; 111 of SEQ ID NO: I or SEQ ID NO: 2. 112 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141 of SEQ ID NO:1 or SEQ ID NO:2; 147 of SEQ ID NO: I, SEQ ID NO:2, or SEQ ID NO:3; 154 of SEQ ID NO: 1 or SEQ ID NO:2: 179 of SEQ ID NO:1, SEQ ID NO: 2. or SEQ ID NO:3; 180 of SEQ ID NO:1 or SEQ ID NO:2: 181 of SEQ IDNO:1 or SEQ ID NO:2; 183 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 184 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 185 of SEQ IDNO:1 or SEQ IDNO:2; 186 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 187 of SEQ ID NO: I or SEQ ID NO: 2. 188 of SEQ ID NO:1 or SEQ ID NO:2; 189 of SEQ ID NO:1 or SEQ ID NO:2; 197 of SEQ ID NO:3; 198 of SEQ ID NO: I or SEQ ID NO:2; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2; 241 of SEQ ID NO:3: 242 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ WO 2016/196344 PCT/US2016/034778 169 ID NO: 1 or SEQ ID NO:2: 247 of SEQ ID NO:3; 248 of SEQ ID NO; 1 or SEQ ID NO:2; 250 of SEQ ID NO:3: 251 of SEQ ID NO:1 or SEQ ID NO:2; 264 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3: 265 of SEQ ID NO: 1 or SEQ ID NO:2; and 286 of SEQ ID NO: 1 or SEQ ID NO:2 . In certain embodiment a s,Shiga, toxin effector polypeptide of the prese ntinvention may comprise one or more of the following amino aci dsubstitutions: KI to A, G, V, L, I, F, M and H; T4 to A, G, V, L, I, F, M, and S; D6 to A, G, V, L, I, F, S, and Q; S8 to A, G, V, I, L, F, and M; T8 to A, G, V, I, L, F, M, and S; T9 to A, G, V, I, L, F, M, and S; S9 to A, G, V, L, I, F, and M; KI 1 to A, G, V, L, I, F, M and H; T12 to A, G, V, 1, L, F, M, and S; S33 to A, G, V, L, I, F, and M; S43 to A, G, V, L, I, F, and M; G44 to A and L; S45 to A, G, V, L, I, F, and M; T45 to A, G, V, L, I, F, and M; G46 to A and P; D47 to A, G, V, L, I, F, S, and Q; N48 to A, G, V, L, and M; L49 to A or G; F50; A51 to V; D53 to A, G, V, L, I, F, S, and Q; V54 to A, G, and L; R55 to A, G, V, L, 1, F, M, Q, S, K, and H; G56 to A and P; 157 to A, G, M, and F; L57 to A, G, M, and F; D58 to A, G, V, L, I, F, S, and Q; P59 to A, G, and F; E60 to A, G, V, L, I, F, S, Q, N, D, M, and R; E61 to A, G, V, L, I, F, S, Q, N, D, M, and R; G62 to A; D94 to A, G, V, L, I, F, S, and Q; R84 to A, G, V, L, I, F, M, Q, S, K, and H; V88 to A and G; 188 to A, G, and V; D94; S96 to A, G, V, I, L, F, and M; T104 to A, G, V, I, L, F, M, and S; A105 to L; T107 to A, G, V, I, L, F, M, and S; S107 to A, G, V, L, I, F, and M; LI08 to A, G, and M; S109 to A, G, V, I, L, F, and M; T109 to A, G, V, 1, L, F, M, and S; G110 to A; Dl l 1 to A, G, V, L, I, F, S, and Q; SI 12 to A, G, V, L, I, F, and M; D141 to A, G, V, L, I, F, S, and Q; G147 to A; VI54 to A and G; RI79 to A, G, V, L, I, F, M, Q, S, K, and H; T180 to A, G, V, L, I, F, M, and S; T181 to A, G, V, L, I, F, M, and S; DI83 to A, G, V, L, I, F, S, and Q; DI84 to A, G, V, L, I, F, S, and Q; L185 to A, G, and V; S186 to A, G, V, I, L, F, and M; G187 to A; R188 to A, G, V, L, I, F, M, Q, S, K, and H; SI89 to A, G, V, I, L, F, and M; DI97 to A, G, V, L, I, F, S, and Q; D198 to A, G. V, L, I, F, S, and Q. R204 to A, G, V, L, I, F, M, Q, S.
K, and H: R205 to A, G, V, L, I, F, M, Q, S, K and H; C242 to A, G, V, and S; S247 to A, G, V, I, L, F, and M; ¥247 to A, G, V, L, I, F, and M; R248 to A, G, V, L, I, F, M, Q, S, K, and H; R250 to A, G, V, L, I, F, M, Q, S, K, and H; R251 to A, G, V, L, I, F, M, Q, S, K, and H; C262 to A, G, V, and S; D264 to A, G, V, L, I, F, S, and Q; G264 to A; and T286 to A, G, V, L, I, F, M, and S, 477. 477. 477. id="p-477" id="p-477" id="p-477" id="p-477" id="p-477" id="p-477" id="p-477" id="p-477" id="p-477" id="p-477"
[477] In addition, any amino acid substitution in one epitope region of a Shiga toxin effector polypeptide which disrupts an epitope while retaining significantShiga toxin effector function is combinabl wite hany other amino aci dsubstituti inon the same or a differ entepitope region which disrupts an epitope while retaining significant Shiga toxin effector function to form a de-immunized, Shiga toxin effector polypepti wideth multiple epitope regions disrupte whiled sti llretaining a significant leve ofl Shiga toxin effector function. In certai embodimentsn a Shiga, toxi effectorn polypept ideof the invention may comprise a combinat ionof two or more of the aforementioned substituti onsand/or the combinati onsof substitutions described in WO 2015/113005. 478. 478. 478. id="p-478" id="p-478" id="p-478" id="p-478" id="p-478" id="p-478" id="p-478" id="p-478" id="p-478" id="p-478"
[478] Base don the empirical evidenc ine the Examples and in WO 2015/113005, certain amino acid regions in the A Subunits of Shiga toxins are predict toed tolerate epitope disruptions while stil retail ning significant Shiga toxi effectorn functions.
For exampl thee, epitope regions natively positioned at 1-15, 39-48, 53-66, 55-66, 94-115, 180-190, 179-190, and 243-257 tolerated multip leamino acid substituti on combinati onssimultaneousl wityhout compromis ingShiga toxin enzymati activityc and cytotoxicity.
B. Examples of Furin-Cleavage Resistant Shiga, Toxin Effector Polypepti des 479. 479. 479. id="p-479" id="p-479" id="p-479" id="p-479" id="p-479" id="p-479" id="p-479" id="p-479" id="p-479" id="p-479"
[479] In certa embodimin ents, the Shig atoxi effen ctor polypept ideof the present invention may compri sea disrupted, furin cleavag motie and/orf furin cleavage site at the carboxy-termi ofnus a Shiga toxi Aln fragment derived region. In certain further embodiments the ,Shiga toxi effen ctor polypepti doesde not comprise any known compensatory structure whic hmay provide furin cleavage proxim toal the carboxy-termi ofnus the Shiga toxi Aln fragment derived region. Non-limiti ng example ofs disrupted furin cleavage motif ands furin cleave sites suitabl fore use in the present invention are described in WO 2015/191764. 480. 480. 480. id="p-480" id="p-480" id="p-480" id="p-480" id="p-480" id="p-480" id="p-480" id="p-480" id="p-480" id="p-480"
[480] Certa furiin n-cleava motge ifdisruptions are indicated herein by reference to specifi aminoc acid positions of native Shig atoxin A Subunits provided in the Sequence Listing, noting that natural occurringly Shiga toxin A Subunits includes precursor form contais ning signa sequencesl of about 22 amino acids at their amino­ terminals which are removed to produce mature Shiga toxi An Subunits and are recogniza toble the skilled worke r.Furthe certair, furin n-cleava motige disrupf tions comprising mutations are indicat hereied byn reference to specific amino acids (e.g.
R for an argini neresidu e)natively present at specifi positc ions within native Shiga toxi An Subunits (e.g. R251 for the arginine residue at position 251 from the amino- -170-WO 2016/196344 PCT/US2016/034778 171 terminus) followed by the amino acid with which that residue has been substituted in tiie particular mutation under discussion (e.g. R251A represents the ammo aci d substitution of alanine for arginine at amino aci dresidue 251 from the amino- terminus). 481. 481. 481. id="p-481" id="p-481" id="p-481" id="p-481" id="p-481" id="p-481" id="p-481" id="p-481" id="p-481" id="p-481"
[481] In certa emboin diments, the Shiga toxin effector polypeptide of the present invention comprises a disrupted funn-cleavage motif at the carboxy-terminus of a Shiga toxin Al fragment derived region, and such embodiments are referre to d herein as "furin-cleava resigestant" or "protease-cle avageresistan" Shigat, toxin effector polypeptides to describe their propertylies relat) iveto wild-type, Shiga toxin A Subunit sand/or wild-type, Shiga toxin Al fragment fusion proteins. 482. 482. 482. id="p-482" id="p-482" id="p-482" id="p-482" id="p-482" id="p-482" id="p-482" id="p-482" id="p-482" id="p-482"
[482] In certain embodiments, the protease-cle avageresistant, Shiga toxin effector polypept ideof the present invention consis essets ntially of a trunca tedShiga toxin A Subunit having two or more mutations. 483. 483. 483. id="p-483" id="p-483" id="p-483" id="p-483" id="p-483" id="p-483" id="p-483" id="p-483" id="p-483" id="p-483"
[483] In certa emboin diments, the protease-cle avageresistant, Shiga toxin effector polypeptide of the present inventi oncompris thees disrupted furin-cleavage motif comprising the amino acid residue substitution (relat iveto a wild-type Shiga toxin polypeptide of one) or both of the arginine residues in the minima l,furin-cleavage site consens motifus with A, G, or H. In certai embodn iment thes, protease-clea vage resistant, Shiga toxin effector polypeptide of the present invention compris aes disruption wrhich compris anes amino acid substitution withi an furin-cleava motifge region, where in the substituti occuron ats the natively positioned amino aci d selected from the group consist ingof: 247 of SEQ ID NOG, 248 of SEQ ID NO: 1 or SEQ ID NOG, 250 of SEQ ID NO:3, 251 of SEQ ID NO: 1 or SEQ ID NOG, or tiie equivalen positt ion in a conserve Shigad toxin effecto polypeptider and/or non- native Shiga toxin effecto polypeptr idesequence In. certain furthe embodiments,r the substitution is to any non-conserva amitiveno acid and tiie substituti occon urs at the native positly ioned amino acid residue position. In certa furthein embodiments,r the mutation compris anes amino acid substitution selected from the group consisting of: R247A, R248A, R250A R25IA, or the equivale positionnt in a conserve Shigad toxin effector polypeptide and/o non-nar tive Shiga toxin effector polypept idesequence. 484. 484. 484. id="p-484" id="p-484" id="p-484" id="p-484" id="p-484" id="p-484" id="p-484" id="p-484" id="p-484" id="p-484"
[484] In certain embodiments, the protease-cle avageresistant, Shiga toxin effector polypeptide of the present invention compris thees disrupted furin-cleavage motif comprising the mutati onwhich is a deletion. In certain further embodiment thes, WO 2016/196344 PCT/US2016/034778 172 disrupt edfurin-cleavage motif compris aes mutation which is a deletion of the region natively positioned at 247-252 in StxA (SEQ II) NO:2) and SLT-1A (SEQ ID NO:3), or the region natively positioned at 246-251 in SLT-2A (SEQ ID NO:3); a deletion of the regio nativelyn positioned at 244-246 in StxA (SEQ ID NO:2) and SLT-1A (SEQ ID NO:3), or the region natively positioned at 243-245 in SLT-2A (SEQ ID NO:3); or a deletion of the region native positly ioned at 253 -259 in StxA (SEQ ID NO:2) and SLT-1A (SEQ ID NO:3), or the region natively positioned at 252-258 in SLT-2A (SEQ ID NO:3). 485. 485. 485. id="p-485" id="p-485" id="p-485" id="p-485" id="p-485" id="p-485" id="p-485" id="p-485" id="p-485" id="p-485"
[485] In certa embodimein nts of the protease-cle avageresistant, Shiga toxin effector polypeptide of the prese ntinvention, the disrupted furin-cleava motifge compris thees mutation which is a carboxy-termina truncal tion as compar toed a wild-type Shiga toxin A Subunit the, truncati whicon hresults in the deletion of one or more amino aci dresidues within the furin-cleava motif.ge In certain further embodiments, the disrupted furin-cleava motifge comprises the carboxy-tennina l truncation which deletes one or more amino acid residues withi then minimal cleava sitge eY/R-x-x-R, such as, e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptide truns, cati endingons at the native aminoly acid residue position 250, 249, 248, 247, 246, 245, 244, 243, 242, 241, 240, or less and; for SLT-2A derived Shiga toxin effector polypeptides trunca, tions ending at the natively amino aci dresidue position 249, 248, 247, 246, 245, 244, 243, 242, 241, or less .Certai n further embodiments compris thee disrupted furin-cleavage motif comprising a combination of any of the aforementioned mutations, where possible. 486. 486. 486. id="p-486" id="p-486" id="p-486" id="p-486" id="p-486" id="p-486" id="p-486" id="p-486" id="p-486" id="p-486"
[486] In certa emboin diments, the disrupt edfurin-cleava motifge compris thees mutation(s) that is a partial, carboxy-tenninal truncation of the furin-cleavage motif: however, certa molecin ules of the present inventi ondo not comprise the disrupt ed furin-cleavage motif which is a complete carboxy-te, rmi truncationnal of the enti re ammo acid residue, furin-cleava motifge For. example, certa in,Shiga toxin effector polypeptides of the present invention comprise the disrupted furin-cleavage motif comprisin a gpartial, carboxy-termi truncanal tion of the Shiga toxin Al fragment regio upn to native position 240 in StxA (SEQ ID NO:2) or SLT-1 A (SEQ ID NO: 1) but not a carboxy-termina trunlcat ation position 239 or less .Similarl y, certai cern, tai Shigan, toxin effector polypepti desof the present invention comprise the disrupt edfurin-cleava motifge comprisin a gparti al,carboxy-termi truncationnal of the Shiga toxin Al fragment region up to native position 239 in SLT-2A (SEQ ID WO 2016/196344 PCT/US2016/034778 173 NO:3) but not a carboxy-termina truncal tion at position 238 or less .In the largest carboxy-termina truncationl of the furm-cleavage resistant, Shiga toxin effector polypeptide of the present invention, mutations comprisin theg disrupte furin-d cleava motif,ge position P14s and P13 of the furin-cleava motifge are still present . 487. 487. 487. id="p-487" id="p-487" id="p-487" id="p-487" id="p-487" id="p-487" id="p-487" id="p-487" id="p-487" id="p-487"
[487] In certa emboin diments, the disrupted furin-cleava motifge compris bothes an amino acid residue substitution within the furin-cleavage motif and a carboxy- terminal truncation as compared to a wild-type, Shiga toxin A Subunit. In certa in furthe embodimer nts, the disrupt edfurin-cleavag motife compris bothes an amino acid residue substitution withi then minimal furm-cleavage site R/Y-x-x-R and a carboxy-termina truncal tion as compared to a wild-type, Shiga toxin A Subunit, such as, e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptides truncat, ions ending at the natively amino acid residue position 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, or great ander comprisi theng natively positioned amino acid residue R248 and/o R25r 1 substitute withd any non-positive charged,ly amino acid residue wher e appropriate and ;for SLT-2A derive Shigad toxin effecto polypeptider truncatios, ns ending at the native aminoly aci dresidue position 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, or great ander comprisin theg natively positione aminod aci dresid ue ¥247 and/o R250r substitut wited hany non-positively charged, amino acid residue where appropria Inte. certain embodiments, the trunca tedShiga toxin effector polypeptide comprisin a gdisrupt edfurin-cleavage motif also comprises the furin- cleava motif,ge amino acid residues at positions P9, P8, and/or P7 in order to maintai optimaln cytotoxicity. 488. 488. 488. id="p-488" id="p-488" id="p-488" id="p-488" id="p-488" id="p-488" id="p-488" id="p-488" id="p-488" id="p-488"
[488] In certa emboin diments, the disrupt edfurin-cleava motifge compris aes mutation(s) whic his one or more internal amino, acid residue deletions, as compared to a wild-type, Shiga, toxin A Subunit. In certa furthein embodiments,r tiie disrupted furin-cleava motifge compris aes mutation which(s) has one or more amino acid residue deletions within the minimal furin-cleavage site R/Y-x-x-R. For example, StxA and SLT-1 A derived Shiga toxin effecto polyper ptide comprisings intern deletionsal of the natively positioned amino acid residues R248 and/or R251, which may be combine witd hdeletions of surroundi resing dues such as, e.g., 249, WO 2016/196344 PCT/US2016/034778 174 250, 247, 252, etc .;and SLT-2A derived Shiga toxin effecto polyper ptide s comprisin interng deletial ons of the natively positioned amino acid residues Y247 and/o R250,r which may be combine witd hdeletions of surroundi resng idues such as, e.g., 248, 249, 246, 251, etc .In certa furtherin embodiment thes, disrupt edfurin- cleava motifge compris aes mutation which is a deletion of four, consecutive amino, aci dresidues which deletes the minimal furin-cleavage site R/Y-x-x-R, such as, e.g., StxA and SLT-1A derived Shiga toxin effector polypepti deslacking R248-R251 and SLT-2A derived Shiga toxin effector polypepti deslacking Y247-R250. In certain furthe embodr iment thes, disrupted furin-cleavage motif compris aes mutation(s) having one or more amino aci dresidue deletions in the amino aci d residues flanking the core furin-cleava motif,ge such as, e.g., a. deletion of 244-247 and/o 252-r 255 in SLT-1A or StxA. In certai furthern embodiment thes, disrupt ed furin-cleavage motif compris aes mutation which is an internal deletion of the enti re surface-expos proteased, e-cle avagesensitive loop as compared to a wild-type, Shiga toxin A Subunit, such as, e.g., for StxA and SLT-IA derived Shiga toxin effector polypeptide a deles, tion of native positly ioned amino acid residues 241-262; and for SLT-2A derived Shiga toxin, effecto polypeptr ides a deletion, of natively positioned amino acid residues 240-261. 489. 489. 489. id="p-489" id="p-489" id="p-489" id="p-489" id="p-489" id="p-489" id="p-489" id="p-489" id="p-489" id="p-489"
[489] In certa emboin diments, the disrupt edfurin-cleava motifge compris bothes a mutation which is an intern aminoal, acid residue deletion within the funn-cleava ge motif and a mutation which is carboxy-termina truncatil ason compared to a wild- type, Shiga toxin A Subunit. In certa furtherin embodiments, the disrupt edfurin- cleava motifge compris bothes a mutation which is an amino acid residue deletion within the minimal funn-cleavage site R/Y-x-x-R and a mutation which is a carboxy-terminal truncation as compared to a wild-type, Shiga, toxin A Subunit. For example, protease-cleava resistant,ge Shiga toxin effecto polypeptidesr may comprise a disrupted furin-cleavage motif comprisi ngmutation(s) which are deletions of the natively positioned amino acid residues 248-249 and/or 250-251 in a truncat StxAed or SLT-1A polypeptide which still has amino acid residue 247 and/o 252,r or the amino acid residues 247-248 and/or 249-250 in a truncate SLT-d 2A whic hstill has amino acid residue 246 and/o 251.r In certa furthein r embodiments, the disrupted furin-cleava motifge comprises a mutati onhaving a deletion of four, consecutive amino, acid residues which deletes the minimal furin- cleava sitege R/Y' -x-x-R and a carboxy-termina truncationl as compared to a wild­WO 2016/196344 PCT/US2016/034778 175 type, Shiga toxin A Subunit, such as, e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptide truncas, tions ending at the native aminly oacid residue position 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, or great ander lacking R248 R25 i: and for SLT-2A derived Shiga toxin effector polypeptides trunca, tions ending at the natively ammo aci dresidue position 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, or great ander lacking Y247-R250.
C. Example ofs Shiga Toxin Effector Polypeptide Havings an Embedde Epitoped 490. 490. 490. id="p-490" id="p-490" id="p-490" id="p-490" id="p-490" id="p-490" id="p-490" id="p-490" id="p-490" id="p-490"
[490] In certa emboin diments, the Shiga toxin effector polypept ideof the present invention may comprise one or more embedded or inserte heted, rologous T-cel, l epitopes for purposes of de-immunizati and/oron delivery7 to a. MHC class I presentation pathway of a targe celt l.For certai embodimen nts and/or certain Shiga toxin effector polypeptide sub-regions, embedding or partia embeddingl a T-cell epitope may be preferr overed inserting a T-cell epitope because, e.g., embedding- type modifications are more likely to be successful in diverse sub-regions of a Shiga toxin effector polypeptide wherea sucs cessfu insel rtions may be more limite tod a smaller subset of Shiga toxin effector polypeptide sub-regions. The term "successful" is used here to mean the modification to the Shiga, toxin effector polypeptide (e.g. introduc tionof a heterologous T-ce,ll epitope) results in a modified Shiga toxin effector polypeptide which retains one or more Shiga toxin effect or functions at the requisite leve ofl activit eity her alone or as a componen oft a. cell - targeting molecule. 491. 491. 491. id="p-491" id="p-491" id="p-491" id="p-491" id="p-491" id="p-491" id="p-491" id="p-491" id="p-491" id="p-491"
[491] Any of tire Shiga toxin effector polypeptide sub-regions describe ind WO 2015/113007 may be suitable for certa embodimin ents of the prese ntinvention, and any of the Shiga toxin effector polypepti desdescribe ind WO 2015/113007 may be modified into a Shiga toxi effen ctor polypeptide of the present invention, e.g., by' die addition of one or more new epitope regio disruptn ions for de-immunization (such one as describe hereid n)and/or a furin-cleava motifge disruption (such as one describe hered in).WO 2016/196344 PCT/US2016/034778 176 492. 492. 492. id="p-492" id="p-492" id="p-492" id="p-492" id="p-492" id="p-492" id="p-492" id="p-492" id="p-492" id="p-492"
[492] In certa emboin diments, the Shiga toxin effector polypeptide of the presen t invention consists essentia oflly a truncat Shigaed toxin A Subuni tcomprising an embedded or inserted hete, rologous T-ce,ll epitope and one or more othe mutations.r In certai embodimen nts, the Shiga toxin, effector polypeptide of the present invention comprises an embedded or inserte heted, rologous T-ce,ll epitope and is smaller than a full-length, Shiga toxin A Subunit, such as, e.g., consist ingof tire polypeptide repres entby amino acid 77s to 239 of SLT-1A (SEQ ID NO: 1) or StxA (SEQ ID NO:2) or die equivale innt othe Ar Subunit sof member ofs the Shiga toxin famil (e.gy .amino acid 77s to 238 of SLT-2A (SEQ ID NO:3)). For example, in certain embodiments of the prese ntinvention, the Shiga toxin effector polypepti des is derived from amino acids 75 to 251 of SEQ ID NO: 1, 1 to 241 of SEQ ID NO: 1, 1 to 251 of SEQ ID NO: 1, or amino acid s1 to 261 of SEQ ID NO: 1, wherei then Shiga toxin effector polypeptide compris ates leas onet embedde ord inserte d, heterologous T-cell epitope and at leas onet amino acid is disrupt edin an endogenous, B-cell and/o CD4+r T-cell epitope region provid edin the Examples (see e.g. Tables 1-7 and/or 12) and wherein the disrupt edamino acid does not overlap with the embedded or inserted epitope. Similarly in othe embodimer nts, the Shiga, toxin effector polypeptide of the present invention is derive fromd amino acid 75s to 251 of SEQ ID NO:2, 1 to 241 of SEQ ID NO:2, 1 to 251 of SEQ ID NO:2, or amino acid s1 to 261 of SEQ ID NO:2, wherein the Shiga toxin effector polypeptide comprises at leas onet embedded or inserte heted, rologous T-cel l epitope and at leas onet ammo acid is disrupt edin an endogenous, B-cell and/o r CD4+ T-cell epitope regio providedn in the Example (sees e.g. Tables 1-7 and/o r 12) and wherein the disrupt edammo acid does not overlap with the embedded or inserted epitope Additionally,. the Shiga toxin effector polypeptide may be derived from amino acid 75s to 251 of SEQ ID NO:3, 1 to 241 of SEQ ID NO:3, 1 to 251 of SEQ ID NO:3, or amino acids I to 261 of SEQ ID NO:3, wherein tire Shiga toxin effector polypeptide compris ates leas onet embedded or inserted hete, rologous T- cell epitope and at leas onet amino acid is disrupted in an endogenous B-cell, and/or CD4+ T-cell epitope regio providedn in the Example (sees e.g. Tables 1-7 and/o r 12) and wherein the disrupt edammo acid does not overlap with the embedded or inserted epitope In. certa embodimin ents of the present invention, the Shiga toxin effector polypeptide compris anes embedded or inserte heterd, ologous, T-cel l epitope and a disrupt edfurin-cleavage motif at the carboxy-termi ofnus a Shiga WO 2016/196344 PCT/US2016/034778 177 toxin Al fragment derived region. For exampl ine certai embodimen nts, the Shiga toxin effector polypeptide of tli prese ent invention is derived from ammo acids 75 to 251 of SEQ ID NO: 1, 1 to 241 of SEQ ID NO: 1, 1 to 251 of SEQ ID NO: 1, or amino acids 1 to 261 of SEQ ID NO: 1, wherei then Shiga toxin effector polypeptide compris ates leas onet embedded or inserted hete, rologous T-cell epitope and a disrupt edfurin-cleavage motif at the carboxy-terminus of a Shiga toxin Al fragment derived region. Similar inly other embodiments, the Shiga toxin effector polypeptide of the present invention is derived from amino acid 75s to 251 of SEQ ID NO:2, 1 to 241 of SEQ ID NO:2, 1 to 251 of SEQ ID NO:2, or ammo acid s1 to 261 of SEQ ID NO:2, where thein Shiga toxin effecto polypeptr idecompris ates leas onet embedded or inserte heted, rologous T-cell epitope and a. disrupted furin- cleava motifge at the carboxy-termi ofnus a Shiga toxin Al fragme derint ved region .
Additiona tirelly, Shiga toxin effector polypeptide may be derive fromd amino acid s 75 to 251 of SEQ ID NO:3, 1 to 241 of SEQ ID NO:3, 1 to 251 of SEQ ID NO:3, or amino acids I to 261 of SEQ ID NO:3, where thein Shiga, toxin effector polypeptide compris ates leas onet embedded or inserted hete, rologous T-cell epitope and a disrupte furin-cld eavage motif at the carboxy-termi ofnus a Shiga toxin Al fragment derived region.
D. Examples of Combinati onShiga Toxin Effector Polypeptides 493. 493. 493. id="p-493" id="p-493" id="p-493" id="p-493" id="p-493" id="p-493" id="p-493" id="p-493" id="p-493" id="p-493"
[493] A combinat ionShiga toxin effector polypeptide of the present invention compris twoes or more sub-regions (?.e. non-overlapping sub-regions) wherei eachn sub-region compris ates leas onet of the following: (1) a disruption in an endogenous epitope or epitope region; (2) an embedded, heterolog T-ceous,ll epitope-peptide (3) an; inserted, heterologous, T-cell epitope-pepti andde; (4) a disrupt edfurin-cleavage motif at the carboxy-termi ofnus an Al fragment derived region. 494. 494. 494. id="p-494" id="p-494" id="p-494" id="p-494" id="p-494" id="p-494" id="p-494" id="p-494" id="p-494" id="p-494"
[494] Certain embodimen ofts the combination Shiga toxin effecto polypeptidesr of the prese ntinvention comprise both (1) a disruption in an endogenous epitope or epitope region and (2) a disrupted furin-cleava motifge at the carboxy-termi ofnus an Al fragment derive regid on. It is predicted that any of the individual, de- immunized, Shiga toxin effector sub-regions described in the Examples belo wor describe ind WO 2015/113005 (see e.g. Table B, supra) may generally be combined with any Shiga toxin effector sub-region comprisin a gdisrupt edfurin-cleavage WO 2016/196344 PCT/US2016/034778 178 motif describe herein,d describe ind WO 2015/191764, and/o knownr in the art in order to create a Shiga toxin effector polypeptide of the prese ntinvention. 495. 495. 495. id="p-495" id="p-495" id="p-495" id="p-495" id="p-495" id="p-495" id="p-495" id="p-495" id="p-495" id="p-495"
[495] In certa embodimin ents of the prese ntinvention, the Shiga toxin effector polypept ideconsis essts ential ofly the polypept ideshown in any one of SEQ ID NOs: 355-438 which furthe comprir ses a disruption of at leas one,t endogenous B- , cell and/or T-cell epitope regio whichn does not overlap with an embedded or inserted, heterologous CD8+, T-cell epitope; wherein the disruption compris onees or more amino acid residue substitutions relat iveto a wild-type Shiga toxin. In certain further embodiments the substitution is selected from the group consist ingof: KI to A, G, V, L, I, F, M and H; T4 to A, G, V, L, I, F, M, and S; D6 to A, G, V, L, I, F, S, Q and R; S8 to A, G, V, I, L, F, and M; T9 to A, G, V, I, L, F, M, and S; S9 to A, G, V, L, I, F, and M; KI 1 to A, G, V, L, I, F, M and H; T12 to A, G, V, I, L, F, M, S, and K; S12 to A, G, V, I, L, F, and M; S33 to A, G, V, L, 1, F, M, and C; S43 to A, G, V, L, I, F, and M; G44 to A or L; S45 to A, G, V, L, I, F, and M; T45 to A, G, N, L, I, F, and M; G46 to A and P; D47 to A, G, V, L, I, F, S, M, and Q; N48 to A, G, V, L, M and F; L49 to A, V, C, and G; ¥49 to A, G, V, L, I, F, M, and T; F50 to A, G, V, L, I, and T; A51 ; D53 to A, G, V, L, I, F, S, and Q; V54 to A, G, I, and L; R55 to A, G, V, L, I, F, M, Q, S, K, and H; G56 to A and P; 157 to A, G, V, and M; L57 to A, V, C, G, M, and F; D58 to A, G, V, L, I, F, S, and Q; P59 to A, G, and F; E60 to A, G, V, L, I, F, S, Q, N, I), M, T, and R; E61 to A, G, V, L, 1, F, S, Q, N, D, M, and R; G62 to A; R84 to A, G, V, L, I, F, M, Q, S, K, and H; V88 to A and G; 188 to A, V, C, and G; D94 to A, G, V, L, I, F, S, and Q; S96 to A, G, V, I, L, F, and M; T104 to A, G, V, L, I, F, M; and N; A105 to L; T107 to A, G, V, L, I, F, M, and P; SI07 to A, G, V, L, I, F, M, and P; LI08 to A, V, C, and G; SI09 to A, G, V, I, L, F, and M; T109 to A, G, V, I, L, F, M, and S; G110 to A; Si 12 to A, G, V, L, I, F, and M; DI 11 to A, G, V, L, I, F, S, Q, and T; S112 to A, G, V, L, I, F, and M; D141 to A, G, V, L, I, F, S, and Q; G147 to A; V154 to A and G. R179 to A, G, V, L, I, F, M, Q, S, K, and H; T180 to A, G, V, L, I, F, M, and S; T181 to A, G. V, L, I, F, M, and S; D183 to A, G, V, L, I, F, S, and Q; DI84 to A, G, V, L, I, F, S, and Q; LI 85 to A, G, V and C; SI86 to A, G, V, I, L, F, and M; G187 to A; RI88 to A, G, V, L, I, F, M, Q, S, K, and H; S189 to A, G, V, I, L, F, and M; DI98 to A, G. V, L, I. F, S, and Q; R204 to A, G, N, L, I, F, M, Q, S, K, and II; R20.5 to A, G, V, L, I, F, M, Q, S, K and H; S247 to A, G, V, I, L, F, and M; ¥247 to A, G, V, L, I, F, and M; R248 to A, G, V, L, I, F, M, Q, S, K, and H; R250 to A, G, V, L, I, F, M, Q, S, K, and H;WO 2016/196344 PCT/US2016/034778 179 R251 to A, G, V, L_ L F. M, 0. S, K, and H; D264 to A, G, V, L, I, F, S. and Q; G264 to A; and T286 to A. G, V, L, I, F, M, and S. In certain further embodiments, there are multiple disruptions of multiple endog, enous B-cell and/or CD8+ T-cell epitope regions wherein each disruption involves at leas onet amino aci dresidue substitution selected from the group consist ingof: KI to A, G, V, L, I, F, M and H; T4 to A, G, V, L, I, F, M, and S; D6 to A, G, V, L, I, F, S, Q and R; S8 to A, G, V, 1, L, F, and M; T9 to A, G, V, I, L, F, M, and S; S9 to A, G, V, L, I, F, and M; KI 1 to A, G, V, L, I, F, M and H; T12 to A, G, V, I, L, F, M, S, and K; S12 to A, G, V, I, L, F, and M; S33 to A, G, V, L, I, F, M, and C; S43 to A, G, V, L, I, F, and M; G44 to A or L; S45 to A, G, V, L, I, F, and M; T45 to A, G, V, L, I, F, and M; G46 to A and P; D47 to A, G, V, L, I, F, S, M, and Q; N48 to A, G, V, L, M and F; L49 to A, V, C, and G; ¥49 to A, G, V, L, I, F, M, and T; F50 to A, G, V, L, I, and T; A51 : D53 to A, G, V, L, I, F, S, and Q; ¥54 to A, G, I, and L; R55 to A, G, V, L, I, F, M, Q, S, K, and H; G56 to A and P; 157 to A, G, V, and M; L57 to A, V, C, G, M, and F; D58 to A, G, V, L, I, F, S, and Q; P59 to A, G, and F; E60 to A, G, V, L, I, F, S, Q, N, D, M, T, and R; E61 to A, G, V, L, I, F, S, Q, N, D, M, and R; G62 to A: R84 to A, G, V, L, I, F, M, Q, S, K and H; V88 to A and G; 188 to A, V, C, and G; D94 to A, G, V, L, I, F, S, and Q; S96 to A, G, V, I, L, F, and M; T104 to A, G, V, L, I, F, M; and N; A105 to L; T107 to A, G, V, L, I, F, M, and P; S107 to A, G, V, L, I, F, M, and P; L108 to A, V, C, and G; S109 to A, G, V, I, L, F, and M; T109 to A, G, V, I, L, F, M, and S; G110 to A; SI 12 to A, G, V, L, I, F, and M; DI 11 to A, G, V, L, I, F, S, Q, and T; SI 12 to A, G, V, L, I, F, and M; DI41 to A, G, V, L, I, F, S, and Q; G147 to A; V154 to A and G. R179 to A, G, V, L, I, F, M, Q, S, K, and H; T180 to A, G, V, L, I, F, M, and S; IT81 to A, G, V, L, 1, F, M, and S; DI83 to A, G, V, L, I, F, S, and Q; DI 84 to A, G, V, L, I, F, S, and Q; L185 to A, G, V and C; S186 to A, G, V, I, L, F, and M; G187 to A; R188 to A, G, V, L, I, F, M, Q, S, K, and H: S189 to A, G, V, I, L, F, and M; DI98 to A, G, V, L, 1, F, S, and Q; R204 to A, G, V, L, I, F, M, Q, S, K, and H: R205 to A, G, V, L, I, F, M, Q, S, K and H; S247 to A, G, V, I, L, F, and M; Y247 to A, G, V, L, I, F, and M; R248 to A, G, V, L, I, F, M, Q, S, K, and H: R250 to A, G, V, L, I, F, M, Q, S, K, and H; R251 to A, G, V, L, I, F, M, Q, S, K, and H; D264 to A, G, V, L, I, F, S, and Q; G264 to A; and T286 to A, G, V, L, I, F, M, and S. 496. 496. 496. id="p-496" id="p-496" id="p-496" id="p-496" id="p-496" id="p-496" id="p-496" id="p-496" id="p-496" id="p-496"
[496] Certa embodin imen ofts the Shiga toxin effecto polypeptidesr of the present invention compris bothe (1) an embedde ord inserte heted, rologous T-ce,ll epitope- WO 2016/196344 PCT/US2016/034778 180 peptide and (2) a disrupt edfurin-cleavage motif at the carboxy-terminus of an Al fragment derived region. Any of the Shiga toxin effector polypept idesub-regions comprising an embedded or inserte heted, rolog T-ceous,ll epitope describe ind the Examples below or in WO 2015/113007 may generall be combiney withd any protease-cle avageresistant, Shiga toxin effector polypeptide sub-region (e.g., modified, Shiga toxin A Subunit sub-regions describe herein,d describe ind WO 2015/191764, and/or known in the art) in order to create a combination, Shiga toxin effector polypeptide which, as a component of a ceil-targeti moleculng ise, both protease-cle avageresistant and capable of deliver inga heterologous T-ce,ll epitope to the MHC class I presenta tionpathway of a target cell Non-l. imiti examng ples of this type of combinat ionShiga toxin, effector polypeptide are shown in SEQ ID NOs: 6-27, 29-32, 340-355, and 370-438. 497. 497. 497. id="p-497" id="p-497" id="p-497" id="p-497" id="p-497" id="p-497" id="p-497" id="p-497" id="p-497" id="p-497"
[497] Certain embodimen ofts the combination Shiga toxin effecto polypeptidesr of the prese ntinvention comprise both (1) a disruptio inn an endogenous epitope or epitope region and (2) an embedded, heterologous T-ce,ll epitope-peptide.
However the, Shiga toxin effecto sub-regionsr comprisin insertg ored embedded, heterologous T-ce,ll epitopes described herein or in WO 2015/191764 are generall y not combinabl withe ever y'de-immunize Shigad, toxin effector sub-regions describe herein,d excep wheret empiricall shown!y to be successfully combined such that the result ingcombination molecule retaine a sufd ficient leve ofl a Shiga toxin effector function(s). The disclos ureherein show's how such embodiments may be made and teste tod empirical demonsly trat succe ess. 498. 498. 498. id="p-498" id="p-498" id="p-498" id="p-498" id="p-498" id="p-498" id="p-498" id="p-498" id="p-498" id="p-498"
[498] The term "successful’’ is used here to mea ntwo or more amino acid residue substitutions in a Shiga toxin effector polypeptide results in a functional feature, such as, e.g., de-immunization, reduced furin-cleavage, and/o abilityr to delive anr embedded or insert epitopeed while, the modified Shiga toxin effector polypeptide retains one or more Shiga toxin effector functions. The approaches and assa ys describe hereid shon w7 how to design, make and empirical tesly embodimet nts of the present invention, which represent combination Shiga, toxin effecto polyper ptide s and cell-targeting molecules comprisin theg same. 499. 499. 499. id="p-499" id="p-499" id="p-499" id="p-499" id="p-499" id="p-499" id="p-499" id="p-499" id="p-499" id="p-499"
[499] lire combination, Shiga toxin effector polypepti desof the prese ntinvention combine the feature ofs their respective sub-region suchs, as, e.g., a furin-cleavage motif disruption, individua epitl ope disruptions, and/o ar heterologous T-cell epitope cargo, and these combinations sometimes resul int Shiga toxin effecto polypeptidesrwit hsynergist reductic ions in immunogenicity as compared to the sum of thei r partiall de-immy unized sub-regions. In particular, the exempla ry,Shiga toxi n effector polypeptide showns in SEQ ID NOs: 13,16 and 21 are synergistical de­ ly immunized due to the combinat ionof two or more sub-regions, one of which comprises an embedded, heterologo T-celus, epitopel and another of whic h comprises an endogen ousepitope disrupt edby one or more amino acid residue substitutions. 500. 500. 500. id="p-500" id="p-500" id="p-500" id="p-500" id="p-500" id="p-500" id="p-500" id="p-500" id="p-500" id="p-500"
[500] For certain embodiments the ,Shig atoxin effector polypeptide of the present invention comprises or consis essentits all of they polypept ideshown in any one of SEQ ID NOs: 6-32, 340-354, and 370-438. For certain embodiments, the combinati on,de-immunized, protease-cleava resigesta Shigant, toxin effector polypepti descomprising embedded, T-cel epitl opes of the present inventio n comprise or consist essentiall of oney of the polypepti desrepresented by SEQ ID NOs: 6-10,13-32, 340-354, and 370-438. [501 ] De-immunized, Shig atoxin effector polypeptide of thes present invention whic hexhibi not cytotoxi orcit reducedy cytotoxici at certty ai concn entrat e.g.ions , Shiga toxin effect polypeor ptide comps rising R179A, may sti llbe useful as de­ immunized, Shiga toxi effen ctor polypeptide for sdelivering exogenous material s into cells. Similarly, CD8+ T-cell hyper-immunize Shigad, toxi effectorn polypeptides of the present invention which exhibi not cytotoxici or tyreduced cytotoxici at certaity concentrn ations e.g. a Shiga, toxin effect polypeptideor comprising an epitope embedded int oits catalytic domain (see e.g. WO 2015/113007, Example 1-F), may stil bel useful for delivering a T-cell epitope(s) to a desired subcellular compartme ofnt a cel inl which the Shiga toxi effen ctor polypept ideis present or as a component of a cell-targeti molngecule for delivery of a T-cell epitope( ints) oa target cell.
E. Exampl esof Cell-Targeti Moleng cule ofs the Present Invention 502. 502. 502. id="p-502" id="p-502" id="p-502" id="p-502" id="p-502" id="p-502" id="p-502" id="p-502" id="p-502" id="p-502"
[502] The Shiga toxi effen ctor polypeptide of thes present inventio mayn be used as components of cell-targeti molngecul thates target various extracel targetlular biomolecules The. following exampl describees in more detai certail structuresn of exemplary cell-tar moleget cule ofs the present inventio whichn target cell s expressi extracelng lulartarget biomolecul suches as, e.g., CD 19, CD20, CD22, CD30, CD38, CD45, HER2, PD-L1, and TYRP1. -181-1. Cell-Targeti Moleng cule Targetis Humng an CD 19 503. 503. 503. id="p-503" id="p-503" id="p-503" id="p-503" id="p-503" id="p-503" id="p-503" id="p-503" id="p-503" id="p-503"
[503] CD 19, also recognized in the art as B4, is a 95 kDa ,B-lineage specific, type- I transmembrane glycoprote presentin on the surface of developing B-cells but not expressed by terminal difflyerent iatplasmed cella s.Whil ethe name CD 19 might refer to multiple proteins wit hrelat structuresed and polypept idesequence froms various species, for the purposes of the structural exampl ofes this section, the term "CD 19" refers to the B-lymphocyt antie gen CD 19 protei presentns in humans whose exact sequence might vary slightly based on the isoform and from individual to individual. With regar tod humans CD19, refers to the prote representedin by the predominant polypeptide sequence UniProt Pl5391 and (National Center 1 Biotechnology Institu te,U.S.) (NCBI) accession AAA69966.1 or AAB60697.1; however, differe isoformnt ands variant exiss duet to splicing, polymorphi sms and/or mutations (see e.g., Kuroki K et al., Genes Immun Suppl 1: S21-30 (2002); Tsuchiya N et al., Arthritis Rheum 50: 4002-7 (2004); Dawidowi Kcz et al., Clin Exp Rheumatol 29: 839-42 (2011)). A skill edworker will be able to identify other CD 19 protei inns humans, even if they diffe fromr the referenced sequences. 504. 504. 504. id="p-504" id="p-504" id="p-504" id="p-504" id="p-504" id="p-504" id="p-504" id="p-504" id="p-504" id="p-504"
[504] CD19 is an attract targetive for targe tedcancer therapie e.g.,s, because of the ubiquitous cell-surface expressi ofon CD 19 by neoplastic cell ands tumors of B-cel l lineages. For example, most malignant B-cel lswere found to expre ssCD 19 (see e.g., Anderson K et al., Blood 63: 1424 (1984); Uckun F et al., Blood 71: 13 (1988); Bradbur Ly et al., J Immunol 149: 2841-50 (1992); Haa sK, Tedder T, Adv Exp Med Biol 560: 125-39 (2005); Tedde T,r Nat Rev Rheumatol 5: 572-7 (2009)). Although CD 19 is considered a pan B-cel marl ker expressed throughou B-cet ll development, mature B-cell ands tumor cell ofs B-cell lineage haves been observed to express three-fold more CD 19 compared to immature B-cells. In particular, CD 19 expressio wasn observed in indolent and aggressi subtyve pes of non-Hodgkin lymphoma (NHL), B-cel chronil lymphocytic leukc emi (B-CLL),a and forms of acut lymphoblase leukemtic ia.Furthermore, due to difference in CDs 19 express ion as compared to CD20 expression, CD19-targeted therapies may be able to target B- cell neoplas atms early stage thans CD2O-target theraed pies. 505. 505. 505. id="p-505" id="p-505" id="p-505" id="p-505" id="p-505" id="p-505" id="p-505" id="p-505" id="p-505" id="p-505"
[505] There are numerous CD19 binding region knowns to the skill edworker whic hmay be associated with a Shig atoxin effect polypeptideor of the present inventio to ncrea tea cell-targeti molecng ule of the prese ntinvention. For purposes of the present invention, the term "CD 19 binding region" refers to a molecular -182-WO 2016/196344 PCT/US2016/034778 183 moiet (e.g.y a proteinace moleous cule or )agent capable of specifica bindinglly an extracellular part of a CL) 19 molecule with high affinity, such as, e.g., having a dissociati constanton with rega rdto CD20 of 105־ to 1012־ mole pers liter As. used herein, CD 19 binding refers to the ability to bind to an extracellu partlar of an isoform or variant of huma nCD 19. 506. 506. 506. id="p-506" id="p-506" id="p-506" id="p-506" id="p-506" id="p-506" id="p-506" id="p-506" id="p-506" id="p-506"
[506] In certa embodimein nts, the CD19 binding region is an immunoglobulin-type binding region. In certa emboin diments, the immunoglobulin-t CD19ype, binding regio isn derived from an immunoglobulin, CD 19 bindin regg ion, such as an antibody paratope capabl ofe bindin gan extracellular part of CD 19. In certain other embodiments, the immunoglobulin-t CDype, 19 binding regio comprin ses an engineered polypept idenot derived from any immunoglobulin domain but which functions like an immunoglobuli CDn, 19 binding regio byn providing high-affini ty binding to an extracel partlular of CD 19. This engineer polypeptideed may optionally include polypeptide scaffolds comprisin org consist ingessentia oflly complementar detery' mining regions and/o antigenr binding regions from immunoglobulins as described herein. 507. 507. 507. id="p-507" id="p-507" id="p-507" id="p-507" id="p-507" id="p-507" id="p-507" id="p-507" id="p-507" id="p-507"
[507] There are numerous CD19 binding regions contemplate as componentsd of the prese ntinvention. Non-limit ingexamples of immunoglobulin-t CD19ype, binding regions include CD19-binding monoclonal antibodies and derivatives thereof, such as humanized variants and recombinant immunoglobuli domains,n e.g., B4 (e.g. clone eB101D3), Leu-12 (Leul2), HD37, B43, CLB-CD19, MOPC 21 components FMC63,, MB19-1, cCD19, B4 89B, SJ25-C1, hA19, huB4, hBU12, XmAb5574, MOR208, MEDI-551, SAR3419, AFM11, GBR 401, XmAb 5871, Hm2E8b, B-l, 5F3, 2E2, 1G9, C-20, F-3, HD237, H-300, M-20, R-20, PDR134, BCE19, HIB19, LE-CD19, LT19, CB19, 6D5, 4G7, AB-1, F974A2, J3-119, MDX- 1342, MAB7489 (clone 771404), and MAB4867 (clon 4G7-2e E3) (see e.g., Caligaris-Ca ppioF et al., J Clin Invest 76: 1243-51 (1985): Chen Z et al., Leuk Res : 1411-7 (1986): Pezzutto A et al., J Immunol 138: 2793-9 (1987): De Rie M et al., LeukRes 12: 135-41 (1988); Uckun F etal BloodIV.., 13-29 (1988); VuistWet al., Cancer Res 49: 3783-8 (1989); Carte Retr al., J Immunol 147: 3663-71 (1991); Zola H et al., Immunol. Cell. Biol 69: 411-22 (1991); Holder M et al., Eur J Immunol 11; 2725-8 (1992); Engel P et al., Immunity 3: 39-50 (1995); Pietersz G et al., Cancer Immunol Immunother 41: 53-60 (1995); Tisone J et al., Am J Clin Pathol 107: 283-91 (1997); WO 2005/012493; Lutz R et al., Proc Am Assoc Cancer Res 47;WO 2016/196344 PCT/US2016/034778 184 3731 (2006): Horton H et al., Cancer Res 68; 8049-57 (2008); Gerbe Hr et al., Blood 113: 4352-61 (2009): Awan F et al., Blood 115: 1204-13 (2010); Herbst Ret al., J Pharmacol Exp Ther 335: 213-22 (2010); Coiffie Br et al., J Clin Oncol 29: 1182-9 (2011); Reusch U et al., Blood 122: 4405 (2013); Breton C et al., JHematol Oncol 7: 33 (2014); Horton H et al., J Immunol 186: 4223-33 (2014); Shen D et al., Monodon Antib Immunodiagn Immunother 33: 215-20 (2014)). Non-limiti ng examples of CD 19 binding regions include scFvs, such as, e.g., FVS191, FVS192, scFv-HD37, scFv-FMC63, HD37-C, HD37-CCH, FMC63-28Z, 4G7mut, 4G7-gra ft (see e.g., Bejeck B et al., Cancer Res 55: 2346-51 (1995); Kipriyanov et al., J Immunol Meth 196: 51-62 (1996); Nichols onI et al., Mol Immunol 34: 1157-65 (1997); WO 2002/050118; Peipp M et al., J Immunol Methods 285: 265-80 (2004); Cheng W et al., Biochim Biophys Acta 1768: 21-9 (2007); Kochenderf J eter al., J Immunother 32: 689-702 (2009); Kugler M et al., Protein Eng Des Sei 22: 135-47 (2009); WO 2012/079000; Kneissi S et al., PLoS One 8: 679047 (2013)). 508. 508. 508. id="p-508" id="p-508" id="p-508" id="p-508" id="p-508" id="p-508" id="p-508" id="p-508" id="p-508" id="p-508"
[508] In certain embodiments, the cell-target moleingcule of the present invention compris aes bindin regiog comprn isi ngan immunoglobulin- polypetype ptide selected, for specifi and.c high-affinity binding to huma nCD 19 and/or the cellul ar surfa ceof a CD 19+ cell In. certain embodiments of the cell-targeting molecule of the prese ntinvention, the binding regio compn rise a spolypeptide( sels)ect fromed the group consist ingof: a) a heavy chain variable (Vh) domain comprisi ng(i) a HABR1 comprising or consisting essenti al ly of one of the amino aci dsequence ass shown in SEQ ID NO:83, SEQ ID NO:89, or SEQ ID NO:96; (ii) aHABR2 comprising or consisting essentially of one of the amino acid sequence as shown in SEQ ID NO: 84, SEQ ID NONO, SEQ ID NO:95, or SEQ ID NO:97; and (iii) a HABR3 comprising or consist ingessentially of one of the amino acid sequenc ase shown in SEQ ID NO:85, SEQ ID NO:91, or SEQ ID NO:98; and b) a light chain variable (Vl) domain comprising (i) a LABRI comprising or consisting essentially of one of the amino acid sequence as shown in SEQ ID NO:86, SEQ ID NO:92, or SEQ ID NO:99; (ii) a LABR2 comprising or consist ingessentially of one of the ammo acid sequenc ase shown in SEQ ID NO:97, SEQ ID NO:93, or SEQ ID NO: 100; and (iii) a LABR3 comprising or consisting essentially of one of the ammo aci dsequenc ase shown in SEQ ID NO:88, SEQ ID NO:94, or SEQ ID NO:101. In certain furthe r embodiments, the cell-targetin molecg ule of the present invention comprise thes bindin gregion comprisin org consist ingessentia oflly amino acid s1-232, 1-233, 1-WO 2016/196344 PCT/US2016/034778 185 234, 1-235, 1-236, 1-242, 1-243, 1-244, 1-245, 1-246, 1-252, 1-253, 1-254, 1- 255, or 1-256 of any one of SEQ ID NOs: 47-119 and 176-248. 509. 509. 509. id="p-509" id="p-509" id="p-509" id="p-509" id="p-509" id="p-509" id="p-509" id="p-509" id="p-509" id="p-509"
[509] According to one specif icbut non-limiti aspngect, the binding regio ofn the cell-target moleculeing of the present invention compris aes ligand (whether naturally occurri orng synthe tic)or a derivative thereof that retains binding functionalit to any extracellular part of CD19. Native CD19 is known to bind at lea stone ligand, CD19-L, a high mobility group (HMG) box protei (seen e.g., Uckun F et al., Br J Haematol 153; 15-23 (2011); US 20120141505). 510. 510. 510. id="p-510" id="p-510" id="p-510" id="p-510" id="p-510" id="p-510" id="p-510" id="p-510" id="p-510" id="p-510"
[510] Any of the aforementioned CD 19 bindin moleculeg mays be suitable for use as a CD 19 binding region or modified to create one or more CD 19 bindin regiog ns for use in a cell-targetin molegcule of the present invention. 2. Cell-Targeti Molngecul Targetinges Human CD20 511. 511. 511. id="p-511" id="p-511" id="p-511" id="p-511" id="p-511" id="p-511" id="p-511" id="p-511" id="p-511" id="p-511"
[511] CD20 (B-lymphoc yteantigen CD20) While the name CD20 might refer to multiple proteins with related structur andes polypeptide sequence froms various species, for the purposes of the structural examples of this section, the term "CD20" refers to the B-lymphocyte antigen CD20 proteins prese ntin humans whose exact sequence might vary7 slightly base don the isoform and from individual to individual.
With rega rdto humans, CD20 refers to the protei repn resente by thed predominan t polypept idesequence UnitPro Plt 1836 and NCB1 accession NP 690605.1; howev er, differ entisoform ands variant exists due to splicing, polymorphisms and/o r mutations (see e.g., Dawidowi Kcz et al., Clin Exp Rheumatol 29; 839-42 (2011); Fang C et al., IntJClin Exp Med 8; 11235-43 (2015)). A skilled worker will be able to identi fyother CD20 proteins in humans even, if they differ from the reference d sequences. 512. 512. 512. id="p-512" id="p-512" id="p-512" id="p-512" id="p-512" id="p-512" id="p-512" id="p-512" id="p-512" id="p-512"
[512] CD20 is a cell-surface glycoprotein expresse byd normal, B-cell lineage cell s withi certan ceinll developmental stage ass well as cel lsof numerous, mature B-cell neoplas ms,such as NHL and chronic lymphocyti leukemic (CLL)a cells. In addition, CD20 is express byed mature T-cell and NK-cell neoplas ms.CD20 is express byed a subset of normal T-cel lsas well as malignant T-cel lssuch as, e.g., in T-cell lymphomas (TCLs) including mycosis fungoides (MF), natural kille celr l lymphom (NK-cea ll lymphoma), peripher T-ceal ll lymphoma (PTCLs),s cutaneous T-cell lymphom as,and T-cell large granula lymphocr yteleukemi (T-LGLL)a . The WO 2016/196344 PCT/US2016/034778 186 association of cell-surfa CD20ce with maligna cellsnt make its an attractive target for cell-targe therapieted s. 513. 513. 513. id="p-513" id="p-513" id="p-513" id="p-513" id="p-513" id="p-513" id="p-513" id="p-513" id="p-513" id="p-513"
[513] There are numerous CD20 binding regions known to the skilled worker which may be associa tedwit ha Shiga, toxin effector polypeptide of the present invention to create a cell-target moleingcule of the present invention. For purposes of the prese ntinvention, the term "CD20 binding region" refers to a molecular moiety (e.g. a proteinace moleous cule or )agent capable of specifica bindinglly an extracellular part of a CD20 molecule wit hhigh affinity, such as, e.g., having a dissociati constanton with rega rdto CD20 of 10-5 to 1012־ mole pers liter As. used herein, CD20 binding refers to the ability׳ to bind to an extracellu partlar of an isoform or variant of huma nCD20. 514. 514. 514. id="p-514" id="p-514" id="p-514" id="p-514" id="p-514" id="p-514" id="p-514" id="p-514" id="p-514" id="p-514"
[514] In certa embodimein nts, the CD20 binding region is an immunoglobulin- type binding region. In certain embodiments, tire immunoglobulin-type, CD20 binding region is derived from, an immunoglobulin, CD20 bindin gregion, such as an antibody paratope capabl ofe bindin gan extracellular part of CD20. In certain other embodiments, the immunoglobulin-t CD20ype, binding region comprises an engineered polypept idenot. derived from any immunoglobulin domain but which functions like an immunoglobuli CD20n, binding regio byn providing high-affini ty binding to an extracel partlular of CD20. This engineer polypeptideed may optionally include polypeptide scaffolds comprisin org consist ingessentia oflly complementa deteryrmin regiing ons and/or antigen binding regions from, immunoglobulins as described herein. 515. 515. 515. id="p-515" id="p-515" id="p-515" id="p-515" id="p-515" id="p-515" id="p-515" id="p-515" id="p-515" id="p-515"
[515] There are numerous CD20 bindin gregions contemplat as componeed ntsof tiie prese ntinvention, such as, e.g. CD20 binding regions described in PCT/US2016/016580. Non-limit ingexamples of immunoglobulin-ty CD20pe, binding regions include monoclonal antibodies and derivatives (e.g., humanized variant ands scFvs) such as, e.g., 1F5, 1H4, 1K1791, 2B8, Leul6 ,Lend, 2F2, 2H7, 7D8, 8E4, 11B8, AME-133v, LY2469298, B9E9, BM-ca, C2B8, CKL GA101, RO5072759, LT20, ibritumom ab,HB20-1-25, MB20-1-18, obinutuzumab, ocaratuzumab, ocrelizumab, PRO70769, ofatumum ab,OUBM1-OUBM8, PRO 131921, rituxima TGLA,b, tositumoma TRU-0b, I5, ublitnximab, veltuznm ab, IMMU-106, hA20, the CD20-binding fibronec domaitin FN3CD20,n and HL23 - scFvs: scFv-1, scFv-3, scFv-5, and scFv-8 (see e.g. Golay J et al., J Immunol 135: 3795-801 (1985); Tedder T et al., Eur J Immunol 16: 881-7 (1986); Liu A et al.,WO 2016/196344 PCT/US2016/034778 187 Proc Natl Acad Sci USA 84: 3439-43 (1987); Press 0 et al., Blood 69: 584-91 (1987): Maloney D et al., Blood 84: 2457-66 (1994): Reff M et al., Blood 83: 435-45 (1994); Hooijberg E et al., Cancer Res 55: 840-6 (1995); Hooijberg E et al., Hybridoma 15: 23-31 (1996); Anderson D et al., Biochem Soo Trans 25: 705-8 (1997); Haisma H et al., Blood 92; 184-90 (1998); Wiseman G et al., Clin Cancer Res 5: 3281s-3286s (1999); Schultz J etal., Cancer Res 60: 6663-9 (2000); Cardarel P etli al., Cancer Immunol Immunother 51: 15-24 (2002); Cheson B, Curr ()pin Investig Drugs 3: 165-70 (2002); Polyak M et al., Blood 99: 3256-62 (2002); Feeling J et al., Blood 104: 1793-800 (2004); Geng S et al., Cell Mol Immunol 3: 439-43 (2006); de Boer O et al., PLoS One 2: 6779 (2007); Burge D et al., Clin Ther : 1806-16 (2008); Hagenbee Ak et al., Blood 111: 5486-95 (2008); Nishida M et aL, Inti J Oncol 31; 1263-74 (2008); Morschha userF et al., J Clin Oncol 27; 3346- 53 (2009); Lim S et al., Haematologica 95; 135-43 (2010); Lv M et al., Cancer Lett 294: 66-73 (2010); Morschhau Fser et al., Ann Oncol 21: 1870-6 (2010); Mdssner E et al., Blood 115: 4393-402 (2010); Olafeso T et al., Protein Eng Des Sei 23; 243-9 (2010); Uchiyama S et al., Cancer Sci 101: 201-9 (2010); Wu L et al., Cancer Lett 292: 208-14 (2010); Alduaij W et al., Blood. 117: 4519-29 (2011); Boross P et al., Haematologica 96: 1822-30 (2011); Fang H et al., Sci China Life Sci 54: 255-62 (2011); Nickerson-Nut C teret al., Rheumatology 50: 1033-44 (2011); Robak T, Robak E, BioDrugs 25: 13-25 (2011); Gang S et al., JHematol Oncol 5: 64 (2012); Sall esG et al., Blood 119: 5126-32 (2012); Abdelwahed R et al., Invest Ophthalmol Vis Sci 54: 3657-65 (2013); Golay J et al., Blood 122: 3482-91 (2013); Kinder M et aL, J Biol Chem 288: 3084-54 (2013); Kobayashi H et al., Cancer Med 2: 130-43 (2013); Natarajan A et al., Clin Cancer Res 19: 6820-9 (2013); Zhang H et al., Cell Physiol Biochem 32: 645-54 (2013); Ahmadzadeh V et ah, Protein Expr Purif 102; 45-41 (2014); Ellbrecht C et al., JAMA Dermatol 1939 (2014); Garff-Tavem Mier et al., Leukemia 28: 230-3 (2014); U.S. patent 4,861,5s 79; 5,500,362; 5,595,721; ,677,180; 5,721,108: 5,736,137; 5,776,456; 5,843,398; 5,849,898; 6,015,542; 6,090,365; 6,120,767; 6,171,586: 6,194,551; 6,224,866; 6,242,195; 6,287,537; 6,306,393; 6,368,596: 6,399,061; 6,410,391; 6,455,043; 6,528,624; 6,538,124; 6,565,827; 6,652,852; 6,682,734; 7,879,984; 8,101,179; 8,153,125; 8,337,844; and patent applicat publicaion tions WO 1995/03770; WO 1998/58964; WO 1999/22764; WO 2000/09160: WO 2000/27428; WO 2000/27433; WO 2000/42072; WO 2000/44788; WO 2000/67795; WO 2000/67796; WO 2000/76542; WO 2001/03734;WO 2016/196344 PCT/US2016/034778 188 WO 2001/10460; WO 2001/10461; WO 2001/10462; WO 2001/13945; WO 2001/72333; WO 2001/80884; WO 2001/97858; WO 2002/060955; WO 2002/079255; WO 2002/096948; WO 2002/102312; WO 2003/002607; WO 2003/061694; WO 2004/032828; WO 2005/000901; WO 2005016969; WO 2006/106959; WO 2009/031230; WO 2014/076292; US 2011/0091483; US 12/0941,583; PCT/US2010/055826; EP20140151932; PCT/GB2012/052532; US 13/048,135; EP20140151932; PCT/GB2012/052532; US 13/048,135; PCT/US2006/046034). 516. 516. 516. id="p-516" id="p-516" id="p-516" id="p-516" id="p-516" id="p-516" id="p-516" id="p-516" id="p-516" id="p-516"
[516] In certa emboin diments, the cell-target moleingcule of the present invention compris aes bindin regiog comprn isi ngan immunoglobulin-type polypeptide selected for specif icand high-affinity bindin tog huma nCD20 and/or the cellul ar surfa ceof a CD20+ cell In. certain embodiments of the cell-targeting molecule of the prese ntinvention, the binding region compris aes polypeptide sel(s)ected from the group consisting of a) a heavy chain variable (Vh) domai comprisingn (i) a HCDR1 comprising or consist ingessentia oflly one of the amino acid sequence ass shown in SEQ ID NO: 102, SEQ ID NO: 108, SEQ ID NO: 114, SEQ ID NO: 120, or SEQ ID NO: 124; (ii) a HCDR2 comprisin org consisting essentia oflly one of the amino acid sequence as shown in SEQ ID NO: 103, SEQ ID NO: 115, or SEQ ID NO: 125; and (iii) aHCDR3 comprising or consist ingessentia oflly one of the amino aci dsequence as shown in SEQ ID NO: 104, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 116, SEQ ID NO: 121, or SEQ ID NO: 126; and b) a light chai variablen (Vl) domain comprisin (i)g a LCDR1 comprisin org consist ingessentiall of oney of tire amino acid sequence as shown in SEQ ID NO: 105, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 117, or SEQ ID NO: 127; (ii) a LCDR2 comprising or consisting essentiall of oney of the ammo acid sequence as shown in SEQ ID NO: 106, SEQ ID NO: 118, SEQ ID NO: 122, or SEQ ID NO: 128; and (iii) a LCDR3 comprising or consisting essentia oflly one of the amino acid sequence as shown SEQ ID NO: 107, SEQ ID NO: 113, SEQ ID NO: 119, SEQ ID NO: 123, or SEQ ID NO: 129. In certa furthein embor diments, the binding regio comprin ses or consists essentially of amino acid s1-245 of any one of SEQ ID NOs: 33, 64, and 65. 517. 517. 517. id="p-517" id="p-517" id="p-517" id="p-517" id="p-517" id="p-517" id="p-517" id="p-517" id="p-517" id="p-517"
[517] Any of the aforementioned CD20 bindin moleculeg mays be suitable for use as a CD20 bindin gregion or modified to create one or more CD20 bindin regiog ns for use in a cell-targetin molegcule of the present invention.WO 2016/196344 PCT/US2016/034778 189 3. Cell-Targeti Molngecul Targetes ingHuman CD22 518. 518. 518. id="p-518" id="p-518" id="p-518" id="p-518" id="p-518" id="p-518" id="p-518" id="p-518" id="p-518" id="p-518"
[518] CD22, als orecogniz ined the art as Siglec- 2,S1GLEC2, BL-CAM, B3, Leu- 14, and Lyb-8, is a transmembr glycoproteinane of abou t120-140 kDa (depending on the spliceofor thatm) binds sial icacid ligands. CD22 is expressed specifica bylly B-celis during developmen andt by a specif icsubse tof mature B-cells. While the name CD22 might refe tor multiple proteins with relate structured ands polypept ide sequenc fromes various species, for the purposes of the structura examl ples of this section, the term "CD22" refe rsto sial icacid-binding lectin proteins prese ntin humans whos exacte sequence might vary slightly base don the isoform and from individua tol individual. With, rega rdto humans, CD22 refe rsto the protei n represented by the predominant polypeptide sequence UniProt P20273 and NCBI accession NP 001265346.1; however, differ entisoform ands variants exist due to splicing, polymorphisms and/o mutatr ions (see e.g., Hitomi Y et al., Tissue Antigens 69: 242-9 (2007); Dawidowic K zet al., Clin Exp Rheumatol 2,9: 839-42 (2011)). A skilled worker will be able to identify other CD22 proteins in humans even, if they differ from the referenced sequences. 519. 519. 519. id="p-519" id="p-519" id="p-519" id="p-519" id="p-519" id="p-519" id="p-519" id="p-519" id="p-519" id="p-519"
[519] As B-cell specif icmarker, CD22 is an attracti targetve for cell-target ed therapies for disease ands conditions involving B-cells, such as, e.g,, conditions involving overactive B-cell eles, vate B-celd populatl ions, B-cell mediated autoimmune diseases, leukemias and, lymphomas (see e.g. Nitschke L, Glycobiology’ 2,4: 807-17 (2014)). In addition, CD22 might be overexpre ssedby a variet ofy malignant B-cells such, as, e.g., B-cell neoplas forms which the majority that have been analyzed expressed cell-surfa CD22.ce 520. 520. 520. id="p-520" id="p-520" id="p-520" id="p-520" id="p-520" id="p-520" id="p-520" id="p-520" id="p-520" id="p-520"
[520] There are numerous CD22 bindin gregions known to the skilled worker which may be associa tedwit ha Shiga, toxin effector polypeptide of the present invention to create a cell-target moleingcule of the present invention. For purposes of the prese ntinvention, the term "CD22 binding region" refers to a molecular moiety (e.g. a proteinace moleous cule or )agent capable of specifica bindinglly an extracellular part of a CD22 molecule with high affinity, such as, e.g., having a dissociati conson tant with rega rdto CD22 of 10° to 10־lz mole pers liter As. used herein, CD2 binding refe rsto the ability to bind to an extracellula part ofr an isoform or varia ofnt human CD 22. 521. 521. 521. id="p-521" id="p-521" id="p-521" id="p-521" id="p-521" id="p-521" id="p-521" id="p-521" id="p-521" id="p-521"
[521] In certa embodimein nts, the CD22 binding region is an immunoglobulin-type binding region. In certain embodiments, the immunoglobulin-type, CD22 binding WO 2016/196344 PCT/US2016/034778 190 regio isn derived from an immunoglobulin, CD22 bindin regg ion, such as an antibody paratope capabl ofe bindin gan extracellular part of CD22. In certain other embodiments, the immunoglobulin-t CD22ype, binding regio comprin ses an engineered polypept idenot derived from any immunoglobulin domain but which functions like an immunoglobuli CD22n, binding regio byn providing high-affini ty binding to an extracel partlular of CD22. This engineer polypeptideed may optionally include polypeptide scaffolds comprisin org consist ingessentia oflly complementa deteryrmin regionsing and/or antige bindingn regions from immunoglobulins as described herein. 522. 522. 522. id="p-522" id="p-522" id="p-522" id="p-522" id="p-522" id="p-522" id="p-522" id="p-522" id="p-522" id="p-522"
[522] There are numerous CD22 binding regions contemplate as componed ntsof the prese ntinvention. Non-lim tingi examples of immunoglobulin-t CD22ype, binding regions include CD22-binding monoclonal antibodies and derivatives thereof, such as humanized variants and recombinant immunoglobuli domains,n e.g., RFB4, alpha S-HCL-1 (alpha Leu-14), HD39, T0L5, 4KB128, HD37, EPB, HD6, LL2, HA22-LR, HB22.7, HulOF4 (MCDT2219A or pinatuzumab), epratuzum ab, inotuzuma CAT-3888b, (BL22), CAT-8015 (moxetumomab and), sc-Fv-4KB128 (see e.g., Campana D et al., J Immunol 134: 1524-30 (1985); Schwarti Rng et al., Blood 65: 974-83 (1985); Darken B et al., J Immunol 136: 4470-9 (1986); Maso nD et al., Blood 69: 836-40 (1987); Ghetie M et al., Cancer Res 48; 2610-7 (1988); Pawlak-Byczkowska E et al., Cancer Res 49: 4568-77 (1989); Press O et al., Cancer Res 49: 4906-12 (1989); Stei nR et al., Cancer Immunol Immunother 37: 293-8 (1993); Leung S et al., Hybridoma 13: 469-76 (1994); WO 1994/027638; Leung S et aL, Mol Immunol 32: 1413-27 (1995); WO 1998/041641; WO 2000/074718; Coleman M et al., Clin Cancer Res 9: 3991S-4S (2003); WO 2003/027135; WO 2003/072036; Arndt M et al., FEES Lett 578: 257-61 (2004); Furma net al., Curr Treat Options Oncol 5: 283-8 (2004); WO 2005/012493; Ho M et al., Proc Natl Acad Sci LISA 103: 9637-42 (2006); U.S. 7,074,403; WO 2008/070569; O’Donne ll et al., Caner Immunol Immunother 58: 1715-22 (2009); Mussai Et al., BrJ Haematol 150: 352-8 (2010); Polson A et al., Leukemia 24: 1566-73 (2010); Wayne et al., Clin Cancer Res 16; 1894-903 (2010); Wong et al., Expert Opin Biol Ther 10: 1251-8 (2010); US 20140248278; JP201518404). 523. 523. 523. id="p-523" id="p-523" id="p-523" id="p-523" id="p-523" id="p-523" id="p-523" id="p-523" id="p-523" id="p-523"
[523] In certa emboin diments, the cell-target moleingcule of the present invention compris aes bindin regiog comprn isi ngan immunoglobulin- polypetype ptide selected for specif icand high-affim bindinty tog huma nCD22 and/or the cellularsurface of a CD22+ cell. In certai embodimn ents of the cell-targeting molecule of the present invention, the binding region comprises a polypeptide(s) selecte fromd the group consisting of: a) a heavy chai varian ble (Vh) domain comprising (i) a HABR1 comprising or consisting essentiall of oney of the amino acid sequences as shown in SEQ ID NO:130, SEQ ID NO: 136, SEQ ID NO: 142, or SEQ ID NO: 148; (ii) a HABR2 comprising or consistin essgentiall of oney of the amino acid sequence as shown in SEQ ID NO: 131, SEQ ID NO:137, SEQ ID NO:143, or SEQ ID NO: 149; and (iii) a HABR3 comprising or consisting essentia oflly one of the amino acid sequence as shown in SEQ ID NO:132, SEQ ID NO:138, SEQ ID NO:144 or SEQ ID NO :150; and b) a light chai varian ble (Vl) domain comprising (i) a LABRI comprising or consisting essentiall of oney of the amm oacid sequenc ase shown in SEQ ID NO: 133, SEQ ID NO: 139, SEQ ID NO: 145, or SEQ ID NO: 151; (ii) a LABR2 comprising or consisti esseng ntiall of oney of the amino aci dsequence as shown in SEQ ID NO: 134, SEQ ID NO: 140, SEQ ID NO: 146, or SEQ ID NO: 152; and (iii) a LABR3 comprising or consisti esseng ntiall of oney of the amino acid sequence as shown in SEQ ID NO:135, SEQ ID NO:141, SEQ ID NO:147, or SEQ ID NO: 153. Alternatively, the binding regions could be describe byd CDRs, which large overlaply wit hABRs and are described in SEQ ID NOs: 154-165. In certa in furthe embodimentsr the ,binding region comprises or consists essentiall of aminoy acids 269-513 of SEQ ID NO: 40 or 80. 524. 524. 524. id="p-524" id="p-524" id="p-524" id="p-524" id="p-524" id="p-524" id="p-524" id="p-524" id="p-524" id="p-524"
[524] Accordi tong one specific but non-limiting aspect, the binding region of the cell-targeti molngecule of the present invention compris aes ligand (whether natural occurrily orng syntheti orc) derivat thereofive that retains binding functionali to CD22,ty such as, e.g., sialic acids, sialic acid-containing glycoconjugates and doma, ins of soluble type-M immunoglobul (IgMsins )(see e.g.
Bakker T et al., Eur J Immunol 32: 1924-32 (2002); Chen W et al., Blood 115: 4778­ 86 (2010); Chen W et al., Leak Lymphoma 53: 208-10 (2012); Schweize Ar et al., Eur J Immunol 42: 2792-802 (2012)). Synthe ticCD22 ligands wit hhigh binding affiniti havees been designed and can be used for cell-targeti (seeng e.g., Razi N, Varki A et al., Proc Natl Acad Set USA 95: 7469-74 (1998); Sliedregt L et al., Bioorg Med Chern 9: 85-97 (2001); van Rossenber S get al., J Biol Chern 216: 12967-73 (2001); Keim S et al., J Exp Med 195: 1207-13 (2002); Collins B et al., J Immunol 177: 2994-3003 (2006); Yu J et al., Biochem Biophys Res Commun 360: 759-64 (2007); Abdu-Alla Hh et al., J Med Chern 51: 6665-81 (2008); O’Reilly M et -191-WO 2016/196344 PCT/US2016/034778 192 al., J Am Chern Soc 130: 7736-45 (2008): Abdu-Aila Hh et al., BioorgMed Chem Lett 19: 5573-5 (2009); Chen W et al., Blood 115: 4778-86 (2010); Lepenies B et al., Curr Opin Chem Biol 14: 404-11 (2010); Abdu-Ailah H et al., Bioorg Med Chem 19: 1966-71 (2011); Chen W et al., Leuk Lymphoma 53: 208-10 (2012); Mesch S et al., ChemMedChem 1; 134-43 (2012); Keim S et aL,Angew Chemlnt Ed Engl 52: 3616-20 (2013); Macauley M et al., J Clin Invest 123: 3074-83 (2013): Preshc Her et al, ACS Chem Biol 9: 1444-50 (2014)). 525. 525. 525. id="p-525" id="p-525" id="p-525" id="p-525" id="p-525" id="p-525" id="p-525" id="p-525" id="p-525" id="p-525"
[525] Any of the aforementioned CD22 bindin moleculeg mays be suitable for use as a CD22 binding region or modified to create one or more CD22 bindin regiog ns for use in a cell-targetin molegcule of the prese ntinvention. 4. Cell-Targeti Moleculng Targetes ingHuman CD30 526. 526. 526. id="p-526" id="p-526" id="p-526" id="p-526" id="p-526" id="p-526" id="p-526" id="p-526" id="p-526" id="p-526"
[526] CD30, als orecogniz ined tire art as tumor necrosis factor recept or superfami 8ly (TNFRSF8) or Ki-1/120, is a type I transmembr glycoproteinane of a size of about 90 to 120 kDa. CD30 functions as a cell-surfa recceeptor (or co- receptor) of the tumor necrosis factor receptor family and binds a ligand, CD30L. A CD30 antigen was first describe asd a marker of classica Hodgkinl lymphoma and Reed-Stemberg cells prese ntin patients with Hodgkin's disease (Schwab U et al, Nature 299: 65-7 (1982); Stein H et ah, Im J Cancer 30: 445-459 (1982)), and CD30 antigen was later observed on non-Hodgkin lymphom cellsa (see e.g. Stein H et ah, Blood 66: 848-58 (1985)). While the name CD30 might refert multipleo proteins wit hrelate structured ands polypeptide sequences from various species, for the purposes of tire structu examplesral of this section, the ter m"CD30" refe rsto the tumor necrosis factor receptor proteins present in humans whose exact sequence might van7 slight basely don the isoform and from individual to individua Withl. rega rdto humans, CD30 refe rsto the protei represn ented by the predominant polypept idesequence UniProt P28908 and NCBI accession AAA51947.1; howev er, different isoform ands varia ntsmay exis duet to splicing, polymorphisms and/o r mutations. A skilled worker will be able to identify othe CD30r proteins in humans , even if they differ from the referenced sequences. 527. 527. 527. id="p-527" id="p-527" id="p-527" id="p-527" id="p-527" id="p-527" id="p-527" id="p-527" id="p-527" id="p-527"
[527] CD30 is an attractive target for cell-target theraed peutic e.g.,s, because its express ionis largely restricted to activat and/ored prolifera lymphocytesting and malignant ceil s.In normal or inflamed tissues, CD30 express ionis largely restric ted to medium/lar activge ated B-cells and/or activated T-cells which produc 1112e -typ eWO 2016/196344 PCT/US2016/034778 193 cytokines (Chiarle R et al., Clin Immunol 90: 157-64 (1990); Werner B et al., J Cutan Pathol 35; 1100-7 (2008); Buchan S, Al-Shamkhani A, PLoS One 7: 645244 (2012)). CD30 is highly express byed certa cellin types such, as, e.g., certain lymphom ceilsa othe, malignar lymphnt oid cell ands, non-lymphoid tumor cell s, whereas only a restrict subsedet of healthy cel lsexpress CD30 and at lower levels (Deutsch Y et al., Leuk Lymphoma 52: 1641-54 (2011)). CD30 is express byed cel ls involved in lymphoprolife disorrativeders lymphoid, neoplasms and, myeloid neoplas ms.For example, CD30 is expressed on a subset of non-Hodgkin lymphoma cells, including Burkitt's ana, plas larticge-c lymphomaell cells (ALCL), T-cell lymphomas, cutaneous T-cell lymphoma cells nodular, small cleaved- celymphomall cells, lymphocyti lymphomac cells peri, phera T-cel ll lymphom cela ls, Lenne’rts lymphom ceilsa imm, unobiast lymphomaic cells T-ce, ll ieukemidymphoma cell s (ATLL), adult T-cell leukemi (T-ALL),a centrobiastic/centrocytic (cb/cc follic) ular lymphom cellsa and, lymphomatoid papulosis cells (see e.g., Stein H et al., Blood 66: 848-58 (1985); Stein et al, Neoplastic Hematophathology, pg 675, (Baltimore, Williams & Wilkins, Knowles D, ed.) (1985); Stein H et ai., Pathology of Cells Receptors and Tumor Markers, pg 121 (Stuttgart Gusta, Fiscv her Verlag, Sefert G, Hubner K (eds) (1987); Suchi T et al., J Clin Pathol 40: 995 (1987); Eckert F et al., A m J Dermatopathol li: 345-52 (1989); Moller P et al., Am J Clin Pathol 91: 18-23 (1989); Bums B, Dardick I, Am J Clm Pathol 93: 327-32 (1990); Pins M et al., Histopathology 17: 211-8 (1990); Miettinen M, Arch Pathol Lab Med 116: 1197- 201 (1992); Norduyn L et al., J Clin Pathol 47: 33-7 (1994); Sabattini E et ai., Haematologica 98: 68 1-2 (2013)). C'D30 express ionhas been observed in embryonal carcinomas, nonembryona carcl inomas malig, nant melanoma s, mesenchyma tumorsl and, myeloid cell lines and macrophages at late stage ofs differentiation (see e.g., Andreesen R et al., Blood 63: 1299-1302 (1984); Schaadt M et al., ini Rev Exp Pathol 27: 185-202 (1985); Stein H et al., Haematol Blood Transfus 29: 441-4 (1985); Froese P et al., J Immunol 139: 2081-7 (1987); Pallese n G, Hamilton-Dut S,oit Am J Pathol 133: 446-50 (1988); Andrees Ren et al., Am J Pathol 134: 187-92 (1989); Hansen H et ai., Biol Chern Hoppe-Seyler 370: 409-16 (1989); Schwarti Rng et al., Blood 74: 1678-89 (1989); Mechtershe imerG, Moll er P, Cancer 66: 1732-7 (1990); Pallesen G, Histopathology 16: 409-13 (1990); Durkop H et al., Cell 68: 421 -7 (1992); Latza U et ai., Am J Pathol 146: 463-71 (1995)). CD30 express ionappea rsupregulated by neoplas masttic cells of advanc edWO 2016/196344 PCT/US2016/034778 194 neoplas ms,such as, neoplas involvedms in mastocytos andis systemic mastocyt osis (see e.g., Soltar K et al., Mod Pathol 24: 585-95 (2011); Vale ntP et al., Leuk Lymphoma 52: 740-4 (2011)). CD30 express ionalso has been reported to increase in a varie tyof autoimmun ande inflammatory' diseases such, as, e.g., lymphoid neoplas ms,myeloid neoplas ms,atopic allergie (atopics dermatiti atopics, asthma, rhinoconjuncti allervitis ,gicrhinitis syst), emic lupus erythematosus, systemic scleros (scisleroderma) graf, t-versus disea-hostse, HI V infection, Epstein-Ban virus infection, measles mononucleos, infecis tion, Omen’s syndrome ulce, rati colitis,ve rheumatoid arthritis mult, iple scleros psoriais, sis Hashim, oto’s thyroiditis, prima ry biliar cirrhosis,y Sjogren’s syndrom toxoplasme, osis,Wegener’s granulomatosi s, and tuberculos (seeis e.g., Ralfkiaer E et al., Arch Dermatol Res 279: 28-292 (1987); Romagnani S et al., J Leukocyte Biol 57: 726-30 (1995); Gruss H et ah, Immunol Today 18: 156-63 (1997); Hone R, Watabab T,e Semin Immunol 10: 457-70 (1998); Bengtsson A, Allergy 561: 593-603 (2001); Gerl Ri et al., Trends Immunol 22: 72-7 (2001)). CD30 express ionis a marker for mastocytos (seeis e.g. Maric J, Calvo K, LeukLymphoma 52: 732-3 (2011)). 528. 528. 528. id="p-528" id="p-528" id="p-528" id="p-528" id="p-528" id="p-528" id="p-528" id="p-528" id="p-528" id="p-528"
[528] There are numerous CD30 binding regions known to the skilled worker which may be associa tedwit ha Shiga, toxin effector polypeptide of the present invention to create a cell-target moleingcule of the present invention. For purposes of the prese ntinvention, the term "CD 30 binding region" refers to a molecular moiety (e.g. a proteinace moleous cule or )agent capable of specifica bindinglly an extracellular part of a CD30 molecule with high affinity, such as, e.g., having a dissociati conson tant with rega rdto CD30 of 10° to 10־lz mole pers liter As. used herein, CD30 binding refers to the ability to bind to an extracellu partlar of an isoform or variant of huma nCD30. 529. 529. 529. id="p-529" id="p-529" id="p-529" id="p-529" id="p-529" id="p-529" id="p-529" id="p-529" id="p-529" id="p-529"
[529] In certa embodimein nts, the CD30 binding region is an immunoglobulin- type binding region. In certain embodiments, tire immunoglobulin-type, CD30 binding region is derived from, an immunoglobulin, CD30 bindin gregion, such as an antibody paratope capabl ofe bindin gan extracellular part of CD30. In certain other embodiments, the immunoglobulin-t CD30ype, binding region comprises an engineered polypept idenot derived from any immunoglobulin domain but which functions like an immunoglobulin, CD30 binding regio byn providing high-affinit y' binding to an extracel partlular of CD30. This engineer polypeptideed may optionally include polypeptide scaffolds comprisin org consist ingessentia oflly WO 2016/196344 PCT/US2016/034778 195 complementa deteryrmin regionsing and/o antiger bindingn regions from immunoglobulins as described herein. 530. 530. 530. id="p-530" id="p-530" id="p-530" id="p-530" id="p-530" id="p-530" id="p-530" id="p-530" id="p-530" id="p-530"
[530] There are numerous CD30 binding regions contemplate as componed ntsof the prese ntinvention. Non-limit ingexamples of immunoglobulin-t CD30ype, binding regions include CD30-binding monoclonal antibodies and derivatives thereof, such as humanized variants and recombinant immunoglobuli domains,n e.g., Ki-1, HeFi-1, Ber-H2 ,Ber-H4, Ber-H6 ,Ber-H8 ,Ber-HlO, HRS-1, HRS-3, HRS-4, AGIO, CIO, Ki-2, Ki-3, Ki-4, Ki-5. Ki-6, Ki-7, M44, M67, scFv-K1-4, scFv 4E3, T6, T7, T13, T14, T21, T24, T25, T104, T105, T107, T112, T20L T214, T215, T405, T406, T408, T411, T420, '1426, '1427 (see e.g., Schwab U et al., Nature 299: 65-7 (1982); Hecht T et al., J Immunol 134: 4231-6 (1985); Schwarting R et al., Issue Sections. In: J. A. McMicha el(ed.) . Leucocyte Typing 3: 574-75. Oxford: Oxford Univers ityPress, (1987); Schwarting R et al., Leucocyte Typing IV; 419-22. Oxford, UK, Oxford Universit (1989);y Bowen M et al., J Immunol 151: 5896-906 (1993); Gruss H et al., Blood 83: 2045-56 (1994); Hom-Lohrens O et al., Int J Cancer 60: 539-44 (1995); WO 1996/022384; Barth S et al., Blood 95: 3909-14 (2000); Klimka A et al., Br J Cancer 83: 252-60 (2000); WO 2002/043661; WO 2003/059282; US 2004/018194; WO 2005/001038; WO 2007/040653; WO 2008/025020; WO 2015/028444). 531. 531. 531. id="p-531" id="p-531" id="p-531" id="p-531" id="p-531" id="p-531" id="p-531" id="p-531" id="p-531" id="p-531"
[531] In certa emboin diments, the cell-target moleculeing of the present invention compris aes bindin regiog comprn isi ngan immunoglobulin- polypetype ptide selected for specif icand high-affinity bindin tog human CD30 and/or the cellul ar surfa ceof a CD30+ cell In. certai embodimen nts of the cell-targeti molecng ule of tiie prese ntinvention, the bindin regg ion comprises a polypeptide selec(s) ted from the group consisting of: a) a heavy chain variable (Vh) domai comprisingn (i) a HABR1 comprising or consist ingessentia oflly one of the amino acid sequence ass shown in SEQ ID NO: 166, SEQ ID NO: 172, SEQ ID NO: 178, or SEQ ID NO: 184; (ii) a HABR2 comprisin org consist ingessentia oflly one of the amino aci dsequence as shown in SEQ ID NO: 167, SEQ ID NO: 173, SEQ ID NO: 179, or SEQ ID NO: 185; and (iii) a HABR3 comprisin org consisting essentia oflly one of the amino acid sequence as shown in SEQ ID NO: 168, SEQ ID NO: 174, or SEQ ID NO: 180; and b) a light chain variable (Vl) domain comprising (i) a LABRI comprising or consisting essen tial lyof one of the amino acid sequence as shown in SEQ ID NO:169, SEQ ID NO:175, SEQ ID NO:181 or SEQ ID NO:186; (ii) aLABR2 WO 2016/196344 PCT/US2016/034778 196 comprising or consist ingessentiall of oney of the amino acid sequence as shown in SEQ ID NO: 170, SEQ ID NO: 176, SEQ ID NO: 182, or SEQ ID NO: 187: and (iii) a LABR3 comprisin org consisting essentia oflly one of the amino acid sequence as shown in SEQ ID NO: 171, SEQ ID NO: 177, SEQ ID NO: 183, or SEQ ID NO: 188.
In certain further embodiments, the cell-target moleculeing of the present invention comprise thes binding region comprisin org consist ingessentia oflly amino acid s 268-500 of any one of SEQ ID NOs: 452, 472, 487, and 503. 532. 532. 532. id="p-532" id="p-532" id="p-532" id="p-532" id="p-532" id="p-532" id="p-532" id="p-532" id="p-532" id="p-532"
[532] According to one specif icbut non-limiti aspngect, the binding region of the cell-target moleculeing of the prese ntinvention compris aes ligand (whether naturally occurri orng synthe tic)or a deri vative thereof that retains binding functionality to an extracellular part of CD30 (see e.g. Powell I et al., JLeukoc Biol 63: 752-7 (1998): Grus sH et al., Eur J Immunol 25: 2083 (1995): Gatt eiV et al., Leuk Lymphoma 35: 21-35 (1999); Zhang P et al., Lab Invest 89: 1423-32 (2009): Parekh P et ah, Biomaterials 34: 8909-17 (2013); Shinod Ka et al., J Autoimmun 57: 14-23 (2015); WO 1993/024135). 533. 533. 533. id="p-533" id="p-533" id="p-533" id="p-533" id="p-533" id="p-533" id="p-533" id="p-533" id="p-533" id="p-533"
[533] Any of the aforementioned CD30 bindin moleculeg mays be suitable for use as a CD30 binding region or modified to create one or more CD30 bindin regiog ns for use in a cell-targetin molegcule of the present invention. 5. Cell-Targeti Molngecul Targetinges Human CD38 534. 534. 534. id="p-534" id="p-534" id="p-534" id="p-534" id="p-534" id="p-534" id="p-534" id="p-534" id="p-534" id="p-534"
[534] CD38 is transmembra proteine charn acte rizedas both a cel surfl ace receptor and extracel lularcycl ADPic ribose hydrolase (ADP-ribosylase) While. the name CD38 might refe tor multiple proteins with related structure ands polypept ide sequence froms various species, for the purposes of the structura examl ples of this section, the term "CD38" refe rsto the cycl ADPic ribose hydrolase proteins present in humans whose exact sequence might vary slightly based on the isoform and from individual to individual. With rega rdto humans CD38 refe rsto the protein represented by the predominant polypeptide sequence UniProt P28907 and NCBI accession BAA 18964; however, differe isoformnt ands variants may exist due to splicing, polymorphi and/osms mutatr ions (see e.g. Ferre roE et al., Immunogenetics 49: 597-604 (1999); Gonzalez-Escriba M noet al., Hum Immunol 65: 660-664 (2004); Drummond F et al., J Bone Miner Metab 24: 28-35 (2006); Aydin S et al, Blood 111: 5646-53 (2008); WO 2006/099875). A skilled worke willr be able to WO 2016/196344 PCT/US2016/034778 197 identify other CD38 proteins in humans, even if they differ from the reference d sequences. 535. 535. 535. id="p-535" id="p-535" id="p-535" id="p-535" id="p-535" id="p-535" id="p-535" id="p-535" id="p-535" id="p-535"
[535] There are numerous CD38 binding regions known to the skilled worker which may be associa tedwit ha Shiga, toxin effector polypeptide of the present invention to create a cell-target moleingcule of the present invention. For purposes of the prese ntinvention, the term "CD 3 8 binding region" refers to a molecular moiety (e.g. a proteinace moleous cule or )agent capable of specifica bindinglly an extracellular part of a CD38 molecule wit hhigh affinity, such as, e.g., having a dissociati constanton with rega rdto CD38 of 10-5 to 1012־ mole pers liter As. used here ,in CD3 8 binding refers to the ability to bind to an extracellu partlar of an isoform or variant of huma nCD38. 536. 536. 536. id="p-536" id="p-536" id="p-536" id="p-536" id="p-536" id="p-536" id="p-536" id="p-536" id="p-536" id="p-536"
[536] In certa embodimein nts, the CD38 binding region is an immunoglobulin- type binding region. In certain embodiments, tire immunoglobulin-type, CD38 binding region is derived from, an immunoglobulin, CD38 bindin gregion, such as an antibody paratope capabl ofe bindin gan extracellular part of CD38. In certain other embodiments, the immunoglobulin-t CD38ype, binding region comprises an engineered polypept idenot. derived from any immunoglobulin domain but which functions like an immunoglobuli CD38n, binding regio byn providing high-affini ty binding to an extracel partlular of CD38. This engineer polypeptideed may optionally include polypeptide scaffolds comprisin org consist ingessentia oflly complementa deteryrmin regiing ons and/or antigen binding regions from, immunoglobulins as described herein. 537. 537. 537. id="p-537" id="p-537" id="p-537" id="p-537" id="p-537" id="p-537" id="p-537" id="p-537" id="p-537" id="p-537"
[537] There are numerous CD38 bindin gregions contemplat as componeed ntsof the prese ntinvention. Non-limiti examplesng of immunoglobulin-t CD38ype, binding regions include CD38-binding monoclonal antibodies and scFvs such as, e.g daratumumab, isatuxima andb, MOR202 (see e.g. Deaglio S et al., Trends Mol Med 14: 210-8 (2008): van de Donk N et al., Immunol Rev 270; 95-112 (2016); WO 1996/016990; WO 2002/006347; WO 2005/103083; WO 2008/047242; WO 2012/092612; WO 2012/092616; US20020164788; US20100285004; US 20150118251). 538. 538. 538. id="p-538" id="p-538" id="p-538" id="p-538" id="p-538" id="p-538" id="p-538" id="p-538" id="p-538" id="p-538"
[538] In certa emboin diments, the cell-target moleingcule of the present invention compris aes bindin regiog comprn isi ngan immunoglobulin- polypetype ptide selected for specif icand high-affinity bindin tog huma nCD38 and/or the cellul ar surfa ceof a CD38+ cell In. certa embodimentsin of tire cell-target moleculeing of WO 2016/196344 PCT/US2016/034778 198 tiie present invention, the bindin regg ion comprises a polypeptide( selecs) ted from tiie group consisting of: a) a heavy chain variable (Vh) domai comprisingn (i) a HABR1 comprising or consist ingessentia oflly one of the amino acid sequence ass shown in SEQ IDNO:189, SEQ TDNO:195, SEQ ID NO:201, SEQ ID NO:207, SEQ ID NO:213, or SEQ ID NO:219; (ii) a HABR2 comprisin org consisting essentia oflly one of tiie ammo acid sequence as shown in SEQ ID NO: 190, SEQ ID NO: 196, SEQ ID NO:202, SEQ ID NO:208, SEQ ID NO:214, or SEQ ID NO:220: and (iii) a HABR3 comprisin org consist ingessentiall of oney of the amino acid sequence as shown in SEQ ID NO: 191, SEQ ID NO: 197, SEQ ID NO:203, SEQ ID NO:209, SEQ ID NO:215, or SEQ ID NO:221; and b) a light chain variable (Vl) domain comprising (i) a LABRI comprisi ngor consist ingessentiall of oney of the amino acid sequence as shown in SEQ ID NO: 192, SEQ ID NO: 198, SEQ ID NO:204, SEQ ID NO:210, SEQ ID NO:216, or SEQ ID NO:222: (ii) a LABR2 comprising or consist ingessentia oflly one of the amino acid sequence as shown in SEQ ID NO: 193, SEQ ID NO: 199, SEQ ID NO:205, SEQ ID NO:211, SEQ ID NO:217, or SEQ ID NO:223; and (iii) a LABR3 comprisin org consist ingessentially of one of the amino acid sequence as shown in SEQ ID NO: 194, SEQ ID NO:200, SEQ ID NO:206, SEQ ID NO:212, SEQ ID NO:218, or SEQ ID NO:224.
Alternatively, the binding regions could be described by CDRs, which largely overlap wit hABRs and are described in SEQ ID NOs: 225-242. In certain further embodiments, the binding region compris ores consists essentia oflly amino acid s 269-499, 269-512, 269-513, or 280-510 of any one of SEQ ID NOs: 34, 35, 41-56, and 82. 539. 539. 539. id="p-539" id="p-539" id="p-539" id="p-539" id="p-539" id="p-539" id="p-539" id="p-539" id="p-539" id="p-539"
[539] A natural CD38 ligand or derivati thereve mayof be utilize asd the binding region of a cell-target moleculeing of the prese ntinvention. Native CD38 is known to bind at least one ligand, CD38L, an Ig protei alsn knowno as platele endothet lial cell adhesion molecule 1 (PECAM1) or CD31 (Cesano A et al., J Immunol. 160: 1106-15 (1998): Deagii oS et al., J Immunol 160: 395-402 (1998)). CD31 or apart of CD31 that interacts with CD38 or a derivative there mayof be fused to Shiga, toxin effector pol ypeptides of the invention to construc CD3t 8-targetin ceil-g, targeting molecules that bind an extracel partlular of CD38. 540. 540. 540. id="p-540" id="p-540" id="p-540" id="p-540" id="p-540" id="p-540" id="p-540" id="p-540" id="p-540" id="p-540"
[540] Any of the aforementioned CD38 binding molecule mays be suitable for use as a CD38 bindin gregion or modified to create one or more CD38 bindin regiog ns for use m a cell-targeti molengcule of the presen invention.tWO 2016/196344 PCT/US2016/034778 199 6. Cell-Targeti Molngecul Targetinges Human CD45 541. 541. 541. id="p-541" id="p-541" id="p-541" id="p-541" id="p-541" id="p-541" id="p-541" id="p-541" id="p-541" id="p-541"
[541] CD45, als orecogniz ined the art as PTPRC (protein tyrosine phosphata se, receptor type, C) and leukocyte common antigen (LCA), is a type I transmembra ne, protein tyros inephosphatas exprese sed on the cell surfaces of many differentia ted hematopoietic cells, especiall maliy gnan hematolt, ogical cells, such as, e.g., lymphoma, B-cell chronic lymphocyti leukemic (B-CLL),a hair cey ll leukemi anda, acute nonlymphocytic leukemia (AML) cells. While the name CD45 might refer to multiple proteins with related structur andes polypeptide sequence froms various species, for the purposes of the structura examl ples of this section, the term "CD45" refers to the protei tyrosinen phosphatas proteinse prese ntin humans whose exact sequence might vary7 slightly base don the isoform and from individual to individual .
With rega rdto humans, CD45 refers to the protei repn resente by thed predominan t polypept idesequence UniProt Q6Q1Q5 and, e.g., NCBI accession NP563578.2 or NP_002829.3; however, differ entisoform ands variants exist due to splicing, polymorphisms and/or mutations (see e.g. Motta-Mena L et al., J Biol Chem 286: 20043-53 (2011); Marme F etal., Breast Cancer Res Treat 132: 819-31 (2012); Pokoyski C et al., Genes Immun 16: 519-27 (2015)). A skilled worker will be able to identi fyother CD45 proteins in humans even, if they differ from the referenced sequences. 542. 542. 542. id="p-542" id="p-542" id="p-542" id="p-542" id="p-542" id="p-542" id="p-542" id="p-542" id="p-542" id="p-542"
[542] There are numerous CD45 bindin gregions known to the skilled worker which may7 be associated with a Shiga toxin effector poly peptide of the present invention to create a cell-target moleingcule of the present invention. For purposes of the prese ntinvention, tire term "CD45 binding region" refers to a molecular moiety (e.g. a proteinace moleous cule or )agent capable of specifica bindinglly an extracellular part of a CD45 molecule with high affinity, such as, e.g., having a dissociati constanton with rega rdto CD45 of 105־ to 1012־ mole pers liter As. used herein, CD45 binding refers to the ability to bind to an extracel partlular of an isoform or variant of hum an CD45. 543. 543. 543. id="p-543" id="p-543" id="p-543" id="p-543" id="p-543" id="p-543" id="p-543" id="p-543" id="p-543" id="p-543"
[543] In certa embodimein nts, the CD45 binding region is an immunoglobulin- type binding region. In certa embodimein nts, the immunoglobulin-type, CD45 binding region is derived from an immunoglobulin, CD45 binding region, such as an antibody paratope capabl ofe bindin gan extracellular part of CD45. In certain other embodiments, the immunoglobulin-t CD45ype, binding regio comprin ses an engineered polypept idenot derived from any7 immunoglobulin domain but which WO 2016/196344 PCT/US2016/034778 200 functions like an immunoglobuli CD45n, binding region by providing high-affini ty binding to an extracel partlular of CD45. This engineer polypeptideed may optionally include polypeptide scaffolds comprisin org consist ingessentia oflly complementa deteryrmin regionsing and/o antir gen binding regions from immunoglobulins as described herein. 544. 544. 544. id="p-544" id="p-544" id="p-544" id="p-544" id="p-544" id="p-544" id="p-544" id="p-544" id="p-544" id="p-544"
[544] There are numerous CD45 bindin gregions contemplat as componeed ntsof the prese ntinvention. Non-limit ingexamples of immunoglobulin-t CD45ype, binding regions include CD45-binding monoclonal antibodies and scFvs such, as, e.g., anti-CD45RB (see e.g., Luke P et al., Cun-Mol Med 1: 533-43 (2001): Lin Y et al., Cancer Res 66: 3884-92 (2006)). 545. 545. 545. id="p-545" id="p-545" id="p-545" id="p-545" id="p-545" id="p-545" id="p-545" id="p-545" id="p-545" id="p-545"
[545] In certain embodiments, the cell-target moleingcule of the present invention compris aes bindin regiog comprn isi ngan immunoglobulin- polypetype ptide selected for specif icand high-affini bindinty tog huma nCD45 and/or the cellul ar surfa ceof a CD45+ cell In. certai embodimen nts of the cell-targeti molecng ule of the prese ntinvention, the bindin regg ion comprises a polypeptide( selecs) ted from tiie group consisting of: a) a heavy chain variable (Vh) domai comprisingn (i) a HABR1 comprising or consist ingessentia oflly one of the amino acid sequence, ass shown in SEQ ID NO:243, SEQ ID NO:249, or SEQ ID NO:255; (ii) a HABR2 comprising or consist ingessentia oflly one of the amino acid sequence as shown in SEQ ID NO:244, SEQ ID NO:250, or SEQ ID NO:256; and (111) a HABR3 comprising or consist ingessentia oflly one of the amino acid sequence as shown in SEQ ID NO:245, SEQ ID NO:25.1, or SEQ ID NO:257; and b) a. light chai nvariable (Vl) domai comprn isin (i)g a LABR I comprising or consisting essentia oflly one of tiie amino acid sequence as shown in SEQ ID NO:246, SEQ ID NO:252, or SEQ ID NO:258; (ii) a LABR2 comprising or consisting essentia oflly one of the amino aci d sequence as shown in SEQ ID NO:247, SEQ ID NO:253, or SEQ ID NO:259; and (iii) a LABR3 comprisi ngor consist ingessentiall of oney of the amino aci d sequence as shown in SEQ ID NO:248, SEQ ID NO:254, or SEQ ID NO:260. 546. 546. 546. id="p-546" id="p-546" id="p-546" id="p-546" id="p-546" id="p-546" id="p-546" id="p-546" id="p-546" id="p-546"
[546] Any of the aforementio CD45ned bindin moleculeg mays be suitable for use as a CD45 bindin gregion or modified to create one or more CD45 bindin regiog ns for use in a cell-targetin molegcule of the prese ntinvention.WO 2016/196344 PCT/US2016/034778 201 7. Cell-Targeti Molngecul Targetes ingHuman HER2 547. 547. 547. id="p-547" id="p-547" id="p-547" id="p-547" id="p-547" id="p-547" id="p-547" id="p-547" id="p-547" id="p-547"
[547] HER2, als orecogniz ined the art as Receptor tyrosine-protei kinasn erbB-e 2, is a transmembr proteinane which functions as a cell suifa.ce receptor for transducing signal acrs oss the cellular membrane to intracellular regulato ofrs cell proliferation and apoptos is.HER2 is also recogniz ined tire art as Neu, erbB-2, pl85, CD340, NGL, and HER2/neu (Coussens L et al., Science 230: 1132-39 (1985); King C et al., Science 229: 974-6 (1985); Semba K et aL, Proc Nad Aead Set USA 82: 6497-501 (1985); Yamamoto T et al.. Nature 319:230-234 (1986), Koka! Y et al., Proc Nad Acad Sc 1 USA 85; 5389-93 (1988); Disis M et al., Cancer Res 54: 16-20 (1994); Yoshino I et al.. J Immunol 152: 2393-400 (1994) see, e.g., GenBank Acc .Nos.
X03363; M17730; NM 004448; SEG_HUMHER20) While the name HER2 might refe tor multiple proteins with related structure ands polypeptide sequence froms various species for, the purposes of the structura examl ples of this section, the ter m ،،HER2" refe rsto the epiderma growthl factor receptor proteins present in humans whose exact sequence might vary7 slightly based on the isoform and from individual to individua Forl. example, HER2 refers to the human protei represn ented by the exempla7 polypeptidery sequences UniProt P04626 and NCBI accessions NP_004439.2, NP_001005862.1, NP_001276865.1, NP_00l276866.1, and NP 001276867.1; however different, isoform ands variant exists due to splicing, polymorphisms and/or mutations (see e.g. Siddig A et al., Ann N TAcad Set 1138: 84-94 (2008); Poole E et al., hu J Uoi Epidemiol. Genet 2: 300-15 (2011); WO 2000/020579). A skilled worker will be able to identify' other HER2 proteins in humans, even if they differ from the referenced sequences. 548. 548. 548. id="p-548" id="p-548" id="p-548" id="p-548" id="p-548" id="p-548" id="p-548" id="p-548" id="p-548" id="p-548"
[548] HER2 is overexpres bysed many cance cellsr notably, breas canct ercell s, and its overexpres ission strongly associa tedwith increas metaed sta incrsis, eas ed disease reoccurrence and poor, prognosis (see e.g. Slamon D et al., Science 235: 177-82 (1987)). 549. 549. 549. id="p-549" id="p-549" id="p-549" id="p-549" id="p-549" id="p-549" id="p-549" id="p-549" id="p-549" id="p-549"
[549] There are numerous HER2 bindin gregions known to the skille workerd which may7 be associated with a Shiga toxin effector polypeptide of the present invention to create a cell-target moleingcule of the present invention. For purposes of the prese ntinvention, tire term "HER2 binding region" refers to a molecular moiety (e.g. a proteinace moleous cule or )agent capable of specifica bindinglly an extracellular part of a HER2 molecule with high affinity, such as, e.g., having a dissociati constanton with rega rdto CD20 of 105־ to 1012־ mole pers liter As. used WO 2016/196344 PCT/US2016/034778 202 herein, HER2 bindin refg ers to the ability to bind to an extracel partlular of an isoform or variant of huma nHER2. 550. 550. 550. id="p-550" id="p-550" id="p-550" id="p-550" id="p-550" id="p-550" id="p-550" id="p-550" id="p-550" id="p-550"
[550] In certa embodimein nts, the HER2 binding regio isn an immunoglobulin- type binding region. In certain embodiments, the immunoglobulin-type, HER2 binding region is derived from an immunoglobuli HER2n, bindin regiong such, as an antibody paratope capable of bindin gan extracellular part of HER2. In certain other embodiments, the immunoglobulin-type, HER2 binding region comprises an engineered polypept idenot derived from any immunoglobulin domain but which functions like an immunoglobuli HER2n, binding region by providing high-affinity binding to an extracel partlular of HER2. 'This engineer polypeptideed may optionally include polypeptide scaffolds comprisin org consist ingessentia oflly complement detearyrmin regionsing and/o antir gen binding regions from immunoglobulins as described herein. 551. 551. 551. id="p-551" id="p-551" id="p-551" id="p-551" id="p-551" id="p-551" id="p-551" id="p-551" id="p-551" id="p-551"
[551] There are numerous HER2 bindin gregions contemplat as componentsed of the prese ntinvention. Non-limit ingexamples of immunoglobulin-t HER2ype, binding regions include HER2-binding monoclonal antibodies and derivatives thereof, such as, e.g., anti-ErbB2, 41)5, 2C4, 7F3, 7C2, mumAb 4D5, chmAb 4D5, (rhu)mAb 4D5, huMAb4D5-L huMAb4D5-2, huMAb4D5-3,11uMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7,huMAb4D5-8, trastuzumab, humanized 520C9,4D5Fc8, hingele rhu4D5,ss non-glycosylate rhu4D5d with mutat edcysteine residues, pertuzumab, and humanize 2C4d (Hudziak R et al., Mol Cell Biol 9: 1165-72 (1989); McKenzie S et al., Oncogene 4:543-8 (1989); Bacus S et O., Molecular Carcinogenesis 3: 350-62 (1990); Hancock M et al., Cancer Res 51: 4575-80 (1991); Maie rL et ah, Cancer Res 51: 5361-5 369 (1991); Stancovski I etaL, Proc Natl Acad Sei USA 88: 8691-5 (1991); TagHabue E et al.. Ini J Cancer 47: 933-937 (1991); Bacus S et al.. Cancer Res 52: 2580-9 (1992); Cart erP et al., Proc Natl Acad Sci USA 89: 4285-89 (1992); Harwerth I et al. J Biol Chern 267: 15160-7 (1992); Kasprzyk P et al. Cancer Res 52: 2771-6 (1992); Lewis G et al., Cancer Immunol Immunother 37: 255-63 (1993); Xu F et af, Int J Cancer 53: 401 -8 (1993); Artea gaC et al., Cancer Res 54: 3758-65 (1994); Shawve Lr et al., Cancer Res 54: 1367-73 (1994), Klapper L et al. Oncogene 14: 2099-109 (1997); WO 1993/21319: WO 1994/00136; WO 1997/00271; WO 1998/77797; US 5,772,997; US 5,783,186, US 5,821,337; US 5,840,525; US 6,949,245; and US 7,625,859).WO 2016/196344 PCT/US2016/034778 203 552. 552. 552. id="p-552" id="p-552" id="p-552" id="p-552" id="p-552" id="p-552" id="p-552" id="p-552" id="p-552" id="p-552"
[552] In certa emboin diments, die cell-target moleingcule of die present invention compris aes bindin regiog comprn isi ngan immunoglobulin-type polypeptide selected for specifi andc high-affinity binding to huma nHER2 and/or the cellu lar surfa ceof a HER2+ cell In. certain embodiments of the cell-targeting molecule of the prese ntinvention, the binding region compris aes polypeptide(s) selected from the group consisting of: a) a heavy chain variable (Vh) domai comprisingn (i) a HABR1 comprising or consist ingessentia oflly one of the amino aci dsequence ass shown in SEQ ID NO:261, SEQ ID NO:268, or SEQ ID NO:274; (ii) a HABR2 comprising or consist ingessentiall of oney of the amino acid sequence as shown in SEQ ID NO:262, SEQ ID NO:269, or SEQ ID NO:275; and (hi) a HABR3 comprising or consist ingessentia oflly one of the amino acid sequence as shown in SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:270, or SEQ ID N():276; and b) a light chain variable (Vl) domai comprn isin (i)g a LABRI comprisin org consisting essentia oflly one of the amino acid sequence as shown in SEQ ID NO: 264, SEQ ID NO:271, or SEQ ID NO:277; (ii) a LABR2 comprisin org consisting essentia oflly one of die amino acid sequence as shown in SEQ ID NO:265, SEQ ID NO:272, or SEQ ID NO: 278; and (iii) a LABR3 comprisin org consist ingessentia oflly one of the amino acid sequence as shown in SEQ ID NO:266, SEQ ID NO:273, or SEQ ID NO:279. Alternati vely,the bindin regionsg coul bed described by CDRs, which largely overlap with ABRs and are described in SEQ ID NOs: 283-303. In other embodiments of the cell-target moleingcule of the present invention, the bindin g regio comprin ses a polypeptide selec(s) ted from the group consist ingof: a) a heavy chain only variab (VlehH) domain comprisin (i)g a HABR1 comprisin org consisting essentiall of they amino aci dsequences as shown in SEQ ID NO:280; (ii) a HABR2 comprisi ngor consisting essentially of the amino acid sequence as shown in SEQ ID NO:281; and (iii) a HABR3 comprisin org consist ingessentia oflly the amino acid sequence as shown in SEQ ID NO:282. In certai furthern embodiments, the bindin regg ion compris ores consists essentia oflly amino acid 269-52s 0 or 269- 521 of any one of SEQ ID NOs: 36, 66, and 67. 553. 553. 553. id="p-553" id="p-553" id="p-553" id="p-553" id="p-553" id="p-553" id="p-553" id="p-553" id="p-553" id="p-553"
[553] A natura ligandl or derivati thereofve may be utilized as the HER2 binding region for a cell-targetin moleculeg of the prese ntinvention. N ative HER2 is known to heterodimeri witze hothe membersr of the ErbB famil upony bindin ligag nds such as epiderma growthl factors like epiregulin and heregulin (Moasser M, Oncogene 26: 6469-87 (2007); Riese D, Cullum R, Semin Cell Dev Biol 2%; 49-56 (2014);WO 2016/196344 PCT/US2016/034778 204 Sollom Jo et al., Cell Signal 26: 70-82 (2014)). ErbB ligands which bind members of the ErbB family include EGF, TGF-a ,amphiregulin, betacellulin, HB-EGF, epireguli HER2-68n, and HER2-100, heregulins herst, atin, NRG-2, NRG-3, and NRG-4 (Justman Q et al., J Biol Chem 277: 20618-24 (2002); Jhabvala-Romero F., et al., Oncogene 22: 8178-86 (2003)). Examples of an ErbB ligand include the heregui ms(HRG), such as the prototype heregulin disclose in dU.S. Patent ,641,869 and Marchionni M et al., Nature 362: 312-8 (1993). Examples of heregul insinclude heregulin heregul-a, ־p1,in heregul־p2in and heregul־p3in (Holmes W et al., Science 256: 1205-10 (1992); US 5,641,869); neu differentia tion factor (NDF) (Pele set al., Cell 69: 205-16 (1992)); acetylcholi recneeptor-induc ing activity (ARIA) (Falls D et al., Cell 72: 801-15 (1993)); glia growthl factors (GGFs) (Marchionni M et al., Nature 362; 312-8 (1993)); sensory and motor neuron derived factor (SMDF) (Ho W et al., J Biol Chem 270: 14523-32 (1995)); y-heregulm (Schaefer G etal., Oncogene 15: 1385-94 (1997)). 554. 554. 554. id="p-554" id="p-554" id="p-554" id="p-554" id="p-554" id="p-554" id="p-554" id="p-554" id="p-554" id="p-554"
[554] An ErbB ligand accordi tong the present inventi onmay also be a synthetic ErbB ligand. The synthetic ligand may be specif icfor a particula ErbBr receptor or , may recogniz partie cula ErbBr receptor complexes. An example of a synthetic ligand is the synthetic heregulin/EGF chimer biregulina (Jones J et ah, FEES Lett, 447: 227-31 (1999)) and the EGF-like domai fragmen HRGpint 177-244. ErbB ligands or a part of an ErbB ligand that interacts wit hHER2 or a derivative there of may be fuse dto Shiga toxin effecto polypeptidesr of the invention to construc t HER2-targeting, cell-targetin molecg ules of the invention that bind an extracellular part of HER2. 555. 555. 555. id="p-555" id="p-555" id="p-555" id="p-555" id="p-555" id="p-555" id="p-555" id="p-555" id="p-555" id="p-555"
[555] Synthe ticpeptides which bind an extracel partlular of HER2 may be utilized as the binding region for targeting. Many peptides have been describe whichd are capable of bindin tog HER2 (see e.g. U.S. patents 5,578,482; 5,856,110; 5,869,445; ,985,553; 6,333,169; 6,987,088; 7,019,017; 7,282,365; 7,306,801; 7,435,797; 7,446,185; 7,449,480: 7,560,111; 7,674,460; 7,815,906, 7,879,325; 7,884,194; 7,993,650; 8,241,630: 8,349,585: 8,389,227; 8,501,909; 8,512,967; 8,652,474; and US 2011/0059090). 556. 556. 556. id="p-556" id="p-556" id="p-556" id="p-556" id="p-556" id="p-556" id="p-556" id="p-556" id="p-556" id="p-556"
[556] In certain embodiments, smal molecl ules which bind an extracellula part ofr HER2 may be utilize asd the bindin gregion for targeting. Man ysmal moleculel s have been describe whicd hare capabl ofe bindin tog HERZ such as tyrosine kinase inhibitors, AZD8931, lapatinib nera, tinib (HKI-272), dacomiti nib(PF-00299804), WO 2016/196344 PCT/US2016/034778 205 afatinib (BIBW 2992) (Barlaam B et al., ACS Med Chern Lett 4; 742-6 (2013); Y u H, Riley G, J Natl Compr Cane Nefw 11: 161-9 (2013); Roskos kiR, Pharmacol Res 87C: 42-59 (2014)). Other smal molecl ules which bind to an extracellular part of HER2 may be identifie usingd methods wel knownl to those of skil inl the art, such as by derivatiz knowning EGFR binders like gefitinib, erlotini AEE788,b, AG1478, AG1571 (SU-5271), AP26113, CO-1686, XL647, vandeta nib,and BMS-690514 (Kurokawa H, Artea gaC, Clin Cancer Res 7: 44368-44428 (2001); Yigitbas Oi et al., Cancer Res 64: 7977-84 (2004); Yu H, Rile yG, J Natl Compr CaneNetw 11: 161-9 (2013); Roskoski R, Pharmacol Res 87C: 42-59 (2014)). 557. 557. 557. id="p-557" id="p-557" id="p-557" id="p-557" id="p-557" id="p-557" id="p-557" id="p-557" id="p-557" id="p-557"
[557] Any of the aforementioned HER2 binding molecules may be suitable for use as a HER2 bindin gregion or modified to create one or more HER2 binding regions for use in a cell-targetin molegcule of the present invention 8. Cell-Targeti Molngecul Targetes ingHuman PD-L1 558. 558. 558. id="p-558" id="p-558" id="p-558" id="p-558" id="p-558" id="p-558" id="p-558" id="p-558" id="p-558" id="p-558"
[558] PD-L1, als orecogniz ined the art as PDL1, programmed cell death 1 ligand 1, PDCD1 Ligand 1, PDCD1L1, PDCD1LG1, B7 homolo 1 g(B7-H1), and CD274, is a ligand for the programmed cel death-l 1 receptor of T- and B-cells (Dong H et a\.,NatMed5; 1365-9 (1999); Freeman G et al., JExpMed 192: 1027-34 (2000); Latchman Y et ak, Nat Immunol 2: 261-8 (2001)) and the B7-1 receptor and CD80 receptor found on T-cel ls(Butte M et al., Immunity 27:111-22 (2007); Park J et al., Blood 116: 1291-8 (2010)). While the name PD-L1 might refert multipleo proteins wit hrelate strd uctu resand polypeptide sequences from various species, for the purposes of the structura examplesl of this section, the ter m"PD-Ll" refers to the PD-1 ligands present in humans whose exact sequence might vary slightly based on the isofor andm from individual to individual. With regar tod humans, PD-Ll refe rs to the protein represented by the predominant polypeptide sequence UniProt Q9NZQ7 or Q9EP73 and NCB1 accession AA113735.1; however, different isofor ms and variant exiss duet to splicing, polymorphisms and/or mutations (see e.g. Abelson A et al., Genes Immun 8: 69-74 (2007); Wang S et al., J Clin Immunol 27: 563-7 (2007); Hayashi M et al., Eur J Endocrinol 158: 817-22 (2008); Mitchell A et ak, J Clin Endocrinol Metab 94: 5139-45 (2009); Yang Q et ak, Clin Exp Rheumatol 29: 13-8 (2011); MaY etak, Im J Clin ExpMed 15: 16585-91 (2015)). A skille d worker will be able to identify other PD-Ll proteins in humans, even if they differ from the reference sequences.dWO 2016/196344 PCT/US2016/034778 206 559. 559. 559. id="p-559" id="p-559" id="p-559" id="p-559" id="p-559" id="p-559" id="p-559" id="p-559" id="p-559" id="p-559"
[559] PD-L1 is absent from most healthy tissue unders normal conditions; however, PD-LI express ioncan be induced by exposur ofe most nuclea ted mammalia cellsn to an interferon(s) (see e.g., Dong H et al., Nat Med 8: 793-800 (2002); Chen L, Nat Rev Immunol 4: 336-47 (2004); Hirano F et al., Cancer Res 65: 1089-96 (2005); Zou W, Chen L, Nat Rev Immunol 8: 467-7 (2008); Flie sD et al., Yale J Biol Med 84: 409-21 (2011); Chen J et al., Immunobiology 217: 385-93 (2012); Spranger S et al. Sei Transl Med 5: 200ral l6(2013)). During certain malignanci PD-Les, 1 upregulation in tumor microenvironment might slead to over - suppression of immune responses to tumor cells, which fit scommon notions abou t PD-LFs involvem inent adaptive immune resistance of tumor cells to a host’s immune system (see e.g., Zou W, Chen L, Nat Rev Immunol 8: 467-7 (2008); Zheng P, Zho Z, Biomark Cancer I; 15-8 (2015)). 560. 560. 560. id="p-560" id="p-560" id="p-560" id="p-560" id="p-560" id="p-560" id="p-560" id="p-560" id="p-560" id="p-560"
[560] PD-LI is an attract targeive fort therapi becauses PD-LIe is strongly express byed certa tumorin cel lsand tumor-infiltra lymphoting cyte whers ea s healthy, human tissues and cells seldom expre sshigh levels of PD-LI at a cellular surfa ce(see e.g. Dong H et al., Nat Med 8: 793-800 (2002); Chen L, Nat Rev Immunol 4: 336-47 (2004); Hirano F et al., Cancer Res 65: 1089-96 (2005); Chen L, Han X, J Clin Invest 125: 3384-91 (2015)). In humans expre, ssion of cell-surfa ce PD-LI by tumor cells has been observe ind a number of primar tumory biopsies and tumor-ce culturedll cells assessed by immunohistochemi includingstry, cells and tissues related to, e.g., carcinomas, glioma B-cells, lymphomas adult, T-cell leukemia/lymphoma (ATLL), angioimmunoblast T-ceicll lymphomas (AITLs), bladder cancers, chronic lymphocyti leukemiac (CLLs)s , epithelial malignanci es, oral squamous cell carcinomas esophageal, squamous cell carcinoma (ESCCss ), lung cancers non-, Hodgki lymphomasn (NHLs), pancrea cancers,tic renal cel l carcinom (RCCs),as sma lllymphocyti lymphoc mas (SLLs), squamous cell carcinom ofas the head and neck (SCCHN), and virus-associate malignand cies(see e.g., Brown J et al., Immunol 170: 1257-66 (2003); Strome S et al., Cancer Res 63: 6501-5 (2003); Wintter etle al., Cancer Res 63: 7462-7 (2003); Thompson R et al., Cancer Res 66: 3381-5 (2006); Nomi T et al., Clin Cancer Res 13; 2151-7 (2007); Thompson et al., Clin Cancer Res 13: 1757-61 (2007); Andorsky D et al., Clin Cancer Res 17: 4232-44 (2011); Chen B et al., Clin Cancer Res 19: 3462-73 (2013); Chen M et al., Oncotarget I; 7913-24 (2016); Wu C et ah, Sci Rep 6: 19740 (2016)).WO 2016/196344 PCT/US2016/034778 207 561. 561. 561. id="p-561" id="p-561" id="p-561" id="p-561" id="p-561" id="p-561" id="p-561" id="p-561" id="p-561" id="p-561"
[561] There are numerous PD-L1 bindin gregions known to the skilled worker which may be associa tedwith a Shiga toxin effector polypeptide of the present invention to create a cell-target moleingcule of the present invention. For purposes of the prese ntinvention, the ter m"PD-Ll bindin gregion" refers to a. molecular moiety (e.g. a proteinace moleous cule or )agent capable of specifica bindinglly an extracellular part of a PD-L1 molecule with high affinity, such as, e.g., having a dissociati constanton wit hregard to PD-L1 of 105־ to 10-12 moles per liter. As used herein, PD-L1 bindin refersg to the ability to bind to an extracellular part of an isoform or variant of human PD-L1. 562. 562. 562. id="p-562" id="p-562" id="p-562" id="p-562" id="p-562" id="p-562" id="p-562" id="p-562" id="p-562" id="p-562"
[562] In certa embodimein nts, the PD-L1 binding region is an immunoglobulin- type binding region. In certain embodiments, the immunoglobulin-type, PD-Ll binding region is derived from an immunoglobuli PD-L1n, binding region, such as an antibody paratope capable of bindin gan extracellular part of PD-Ll. In certa in other embodiments, the immunoglobulin-t PD-Llype, bindin gregio comprisn anes engineered polypept idenot derive fromd any immunoglobulin domain but which functions like an immunoglobuli PD-Lln, bindin gregion by providing high-affinity binding to an extracellular part of PD-Ll. This engineer polypeptideed may optionally include polypeptide scaffolds comprisin org consist ingessentia oflly complement detearyrmin regionsing and/o antir gen binding regions from immunoglobulins as described herein. 563. 563. 563. id="p-563" id="p-563" id="p-563" id="p-563" id="p-563" id="p-563" id="p-563" id="p-563" id="p-563" id="p-563"
[563] There are numerous PD-Ll bindin gregions contemplat as componeed ntsof the prese ntinvention. Non-limit ingexamples of immunoglobulin-t PD-Llype, bindin gregions include numerous antibodies and immunoglobulin domains already known to bind an extracellula part ofr PD-Ll prese ntat a cellular surface include MDX-1105, MOM-18534-S(P), and various scFvs (see e.g., Latchman Y et al., Nat Immunol 2: 261-8 (2001): Xern L et al., Human Pathol 39: 1050-8 (2008); Chen B et al., Clin Cancer Res 19: 3462-73 (2013): Drees J et al., Protein Expr PurifW 60- 6 (2014): WO 2007/005874: WO 2010/036959: WO 2013/019906; US 7,943,743; US 8,552,154; US 9,102,727; US 9,273,135, and U.S. pate ntapplications US20110271358 and US20160075782). 564. 564. 564. id="p-564" id="p-564" id="p-564" id="p-564" id="p-564" id="p-564" id="p-564" id="p-564" id="p-564" id="p-564"
[564] In certa emboin diments, the cell-target moleingcule of the present invention compris aes binding regio comprn isi ngan immunoglobulin- polypetype ptide selected for specif icand high-affinity bindin tog huma nPD-Ll and/o ther cellul ar surfa ceof a PD-Ll+ cell In. certai embodimen nts of the cell-targetin molecg ule of WO 2016/196344 PCT/US2016/034778 208 tiie present invention, the bindin regg ion comprises a polypeptide selec(s) ted from tiie group consisting of: a) a heavy chain variable (Vh) domai comprisingn (i) a HCDR1 comprising or consist ingessentia oflly one of the amino acid sequence ass shown in SEQ ID NO:304, SEQ ID XOv B!. SEQ ID NO:316, SEQ ID NO:322, or SEQ ID NO:328; (ii) a HCDR2 comprisin org consisting essentia oflly one of the amino acid sequence as shown in S SEQ ID NO:305, SEQ ID NO:311, SEQ ID NO:317, SEQ ID NO:323, or SEQ ID NO:329; and (iii) aHCDR3 comprisin org consisting essentiall of oney of the amino acid sequence as shown in SEQ ID NO:306, SEQ ID NO:312, SEQ ID NO:318, SEQ ID NO:324, or SEQ ID NO:330; and b) a light chain variable (Vl) domain comprisin (i)g a LCDR1 comprising or consisting essentiall of oney of the ammo acid sequence as shown in SEQ ID NO:307, SEQ ID NO:313, SEQ ID NO:319, SEQ ID NO:325, or SEQ ID NO;331; (ii) a LCDR2 comprisin org consisting essential ofly one of the amino acid sequence as shown in SEQ ID NO:308, SEQ ID NO:314, SEQ ID NO:320, SEQ ID NO:326, or SEQ ID NO:332;and (iii) a LCDR3 comprisin org consist ingessentiall of oney of tiie amino acid sequence as shown in SEQ ID NO:309, SEQ ID NO:315, SEQ ID NO:321, SEQ ID NO:327, or SEQ ID NO:333. In certain further embodiments, the bindin gregion compris ores consists essentiall of aminoy acids 269-498 or 269-499 of any one of SEQ IDNOs: 37-39, 68-79, and 81. 565. 565. 565. id="p-565" id="p-565" id="p-565" id="p-565" id="p-565" id="p-565" id="p-565" id="p-565" id="p-565" id="p-565"
[565] Any of tiie aforementioned PD-L1 bindin molecg ules may be suitable for use as a PD-L1 bindin gregion or modified to create one or more PD-L1 binding regio ns for use in a cell-targetin molegcule of the prese ntinvention.
Other Structura Varial tions 566. 566. 566. id="p-566" id="p-566" id="p-566" id="p-566" id="p-566" id="p-566" id="p-566" id="p-566" id="p-566" id="p-566"
[566] In certain embodiments of the cell-targeti moleculeng ofs the present invention, the bindin regg ion has been previously described, e.g., in WO 2005/092917, WO 2007/033497, US2009/0156417, JP4339511, EP1727827, DE602004027168, EPI945660, JP4934761, EP2228383, US2013/0196928, WO 2014/164680, WO 2014/164693, WO 2015/138435, WO 2015/138452, WO 2015/113005, WO 2015/113007, WO 2015/191764, US20150259428, 62/168,758, 62/168,759, 62/168,760, 62/168,761, 62/168,762, 62/168,763, and PCT/US2016/016580. 567. 567. 567. id="p-567" id="p-567" id="p-567" id="p-567" id="p-567" id="p-567" id="p-567" id="p-567" id="p-567" id="p-567"
[567] In certain further embodimen ofts the cell-target moleculeing of the present invention, tire bindin regg ion compris aes polypeptide( selecs) ted from the group WO 2016/196344 PCT/US2016/034778 209 consisting of: (a) a heavy chain variable (Vh) domain comprisin a gHCDR1 comprising or consisting essentiall of they amino acid sequences as shown in SEQ ID NO:334, aHCDR2 comprising or consist ingessentially of the amino acid sequenc ases shown in HCDR2, and a HCDR3 comprisin org consist ingessentially of the amino acid sequence ass shown in SEQ ID NO:336; and (b) a light chain variable (Vl) domain comprising a LCDRl comprisin org consist ingessentia oflly the amino aci dsequenc ases shown in SEQ ID NO:337, a LCDR2 comprising or consisting essentiall of they amino acid sequences as shown in SEQ ID NO:338, and a LCDR3 comprisi ngor consisting essentiall of they ammo acid sequence ass shown in SEQ ID NO:339. 568. 568. 568. id="p-568" id="p-568" id="p-568" id="p-568" id="p-568" id="p-568" id="p-568" id="p-568" id="p-568" id="p-568"
[568] It is within the scope of the prese ntinvention to use fragments, variants , and/o derir vati ofves the cell-targeti molecng ules of the prese ntinvention which contain a functiona bindingl site to any extracellula part ofr a targ biomolecule,et and even more preferabl capyable of bindin ga targe biomoleculet with high affinity (e.g. as shown by Kd). For example, any bindin gregion which binds an extracellular part of a targe biomolecult withe a dissociation constant (Kd) of 105־ to -12 moles/lit prefer, erab lessly than 200 nM, may be substitute ford use in making cell-targetin molecg ules of the invention and methods of the invention. 569. 569. 569. id="p-569" id="p-569" id="p-569" id="p-569" id="p-569" id="p-569" id="p-569" id="p-569" id="p-569" id="p-569"
[569] The skilled worker will recogniz thate variati onsmay be made to the Shiga toxin effector polypepti desand cell-targeting molecules of the present invention, and polynucleoti encodingdes any of the former, without diminishi ngthei biologicar l activities, e.g., by maintaining the overall structure and functi onof the Shiga toxin effector polypeptide such, as in conjunction with one or more 1) endogenous epitope disruptions which reduce antigenic and/or immunogenic potentia 2) l,furin-cleava ge motif disruptions w'hich reduce proteolytic cleavage and/o, 3)r embedded or insert ed epitopes which reduce antigenic and/o immunor geni potentiac or arel capable of being deliver toed a MHCI molecule for presentat onion a cell surface. For example, some modificatio mayns facilit expression,ate facilit purificate ation, improve pharmacokinetic propert iesand/or, improve immunogenici Suchty. modifications are well known to the skilled worker and include, for exampl ae, methionine added at the amino-term inusto provide an initiation site, additional amino acids placed on either terminus to create conveniently locate resd tricti siteson or terminat codoion ns, and biochemica affinil tagsty fused to either terminus to provide for convenient detect ionand/or purification. A common modification to WO 2016/196344 PCT/US2016/034778 210 improve the immunogenicit of ay polypeptide produced using a non-chordat sysetem (e.g. a prokaryoti cell)c is to remov afte, erthe production of the polypeptide, the starti methionineng residue, which may be formyla duringted production, such as, e.g., in a bacterial host system becaus, e,e.g., the presence of N-formylmethionine (fMet) might induc eundesirabl immunee response in schordates. 570. 570. 570. id="p-570" id="p-570" id="p-570" id="p-570" id="p-570" id="p-570" id="p-570" id="p-570" id="p-570" id="p-570"
[570] Also contemplat hereined is the inclusion of additiona aminol acid residues at the amino and/or carboxy termini of a Shiga toxin effector polypeptide of the present invention, a cell-targetin molegcule of the prese ntinvention, or a proteinaceous component of a cell-targeting molecules of the present invention, such as sequence fors epitope tags or othe moietr ies The. additional amino acid residues may be used for various purposes including, e.g., facilitatin clonig ng, facilitating expression, post-translation modifalicat ion,facilitatin syntheg sis,purification, facilitatin detecg tion, and administration. Non-limiti examplesng of epitope tags and moieti esare chitin bindin gprotei domains,n enteropepti cleadasevage sites , Factor Xa cleava sitgees, FIAsH tags, FLAG tags, green fluorescent proteins (GFP), glutathione-S-transf moieterasies HAe, tags, malto bindingse protei domains,n myo tags polyhisti, dine tags, ReAsH tags, strep-tags, strep-tag II, TEV protea sitesse , thioredoxin domains, thrombi clean vage site, and V5 epitope tags. 571. 571. 571. id="p-571" id="p-571" id="p-571" id="p-571" id="p-571" id="p-571" id="p-571" id="p-571" id="p-571" id="p-571"
[571] In certa ofin the above embodiments, the polypeptide sequence of the Shiga toxin effector polypepti desand/o cellr -targetin molecg ules of the present invention are varied by one or more conservati amiveno aci dsubstituti onsintroduced into the polypept ideregion(s) as long as all required structural feature ares still present and tire Shiga toxin effector polypept ideis capable of exhibiting any required function(s), either alone or as a component of a cell-targeti molecng ule. As used herein, the ter m"conserva substtive itution'־' denotes that one or more amino acids are replaced by another, biologica simllyila amir no acid residue. Examples include substitution of amino aci dresidues with simila charactr erist e.g. ics,smal ammol acids aci, dic amino acids, pola aminor acids basi, camino acids, hydrophobic amino acid ands aromat amiic no acid (ses e, for example, Table C). An example of a conservati substive tution with a residue normally not found in endogenous , mammalian peptide ands proteins is the conserva substtive ituti ofon an arginin ore lysine residue with for, exampl ornite, hine, canavanine amino, ethylcys ortei ne, another basic amino acid .For furthe inforr mati conceron ning phenotypic silallyent WO 2016/196344 PCT/US2016/034778 211 substitutions in peptides and proteins see, e.g., Bowie J et al.. Science 247; 1306-10 (1990).
TABLE C. Examples of Conservative Amino Acid Substitutions I II III IV V VI VII VIII IX X XI xn xm XIV A D H F N A F A A A D c c C G E I W G M H C D C C E Q P R L Y S I P W F E D D G Q S N M T L Y G H G E K T H K N P V V G I N P H Q L S K R Q s M R T N R S V T Q T T R V S W P Y T 572. 572. 572. id="p-572" id="p-572" id="p-572" id="p-572" id="p-572" id="p-572" id="p-572" id="p-572" id="p-572" id="p-572"
[572] In the conservative substitution schem me Table C, exempla consry erva tive substitutions of amino acid ares grouped by physicochem properical ties - I; neutr al, hydrophil II:ic; acid ands amides; III: basic; IV: hydropho bic;V: aromatic bulky, amino acids VI, hydrophil uncharged,ic VII aliphatic uncharged, VIII non-pola r uncharged, IX cycloalkenyl-associ X hydrophated, obic, XI polar, XII small, XIII turn-permitt anding, XIV flexible For. example, conservati aminove acid substitutions include the following: 1) S may be substitute ford C; 2) M or L may be substitute ford F; 3) Y may be substitute ford M; 4) Q or E may be substituted for K; ) N or Q may be substitute ford H; and 6) H may be substitute ford N. 573. 573. 573. id="p-573" id="p-573" id="p-573" id="p-573" id="p-573" id="p-573" id="p-573" id="p-573" id="p-573" id="p-573"
[573] Additional conservati aminove acid substitutions include the following: 1) S may be substituted for C; 2) M or L may be substituted for F; 3) Y may be substituted for M; 4) Q or E may be substitute ford K; 5) N or Q may be substituted for H; and 6) H may be substituted for N. 574. 574. 574. id="p-574" id="p-574" id="p-574" id="p-574" id="p-574" id="p-574" id="p-574" id="p-574" id="p-574" id="p-574"
[574] In certa embodimein nts, the Shiga toxin effecto polypeptidesr and cell- targeting molecule ofs the prese ntinvention may compris funce tiona fralgmen orts variant of sa. polypeptide region of the present inventi ondescribed here inthat have, at most, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to a polypeptide sequence recited herein, as long as it (1) compris ates leas onet embedde ord inserte heterologousd, T-cell epitope and at leas onet amino aci dis disrupted in an endogenous, B-cell and/or CD4+ T-cell epitope regio providedn in die Example (sees e.g. Tables 1-7 and/o 12),r wherein the disrupted amino acid does WO 2016/196344 PCT/US2016/034778 212 not overlap with the embedded or inserted epitope; (2) compris ates leas onet embedded or inserte heterologousd, T-cell epitope and a disrupt edfurin-cleavage motif at the carboxy-terminus of a Shiga toxin Al fragment derived region; or (3) compris a.es disrupted furin-cleava motifge at the carboxy-terminus of a Shiga toxin A1 fragment derived region and comprises at least one amino acid is disrupt edin an endogenous, B-cell and/o CD4+r T-cell epitope region provide md the Examples (see e.g. Tables 1-7 and/or 12), wherei then disrupt edamino acid does not overlap wit hthe disrupt edfurin-cleavage motif. Variants of die Shiga toxin effector polypepti desand cell-targeti molecng ules of the invention are withi dien scope of the prese ntinvention as a result of changing a polypeptide described herei byn altering one or more amino aci dresidues or deleting or inserting one or more amino acid residues such, as withi then bindin gregion or Shiga toxin effector polypept ide region, in order to achie vedesired propert iessuch, as changed cytotoxicity, changed cytostatic effects cha, nged immunogenicity, and/o char nged serum half-lif Thee.
Shiga toxin effector polypepti desand cell-targeti molecng ules of the present invention may furthe ber with or without a signal sequence. 575. 575. 575. id="p-575" id="p-575" id="p-575" id="p-575" id="p-575" id="p-575" id="p-575" id="p-575" id="p-575" id="p-575"
[575] Accordingly, in certa embodimein nts, the Shiga toxin effecto polyper ptide s of the prese ntinvention comprise or consists essentia oflly amino acid sequence s having at leas 55%,t 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%, overa sequell nce identit toy a natura occurrilly Shigang toxin A Subunit or fragment there of,such as, e.g., Shiga toxin A Subunit, such as SLT-1A (SEQ ID NO: 1), StxA (SEQ ID NO:2), and/or SLT-2A (SEQ ID NO:3), wherein the Shiga toxin effector polypeptide (1) compris ates leas onet embedded or inserted , heterologous T-cell epitope and at lea stone amino acid is disrupt edin an endogenous, B-cell and/o CD4+r T-cell epitope region provide ind the Examples (see e.g. Tables 1-7 and/or 12), and wherein the disrupt edamino acid does not overlap with the embedded or insert epitope;ed (2) compris ates leas onet embedde d or inserte heted, rologous T-cell epitope and a disrupt edfurin-cleavage motif at the carboxy-terminus of a Shiga toxin Al fragment derived region; or (3) comprises a. disrupt edfurin-cleavage motif at the carboxy-terminus of a Shiga toxin Al fragment derived regio andn compris ates leas onet amino acid is disrupted in an endogenou s, B-cell and/o CD4+r T-cell epitope region provided in the Examples (see e.g. Tables 1-7 and/o 12),r and wherein the disrupt edamino acid does not overlap with the disrupt edfurin-cleavage motif.WO 2016/196344 PCT/US2016/034778 213 576. 576. 576. id="p-576" id="p-576" id="p-576" id="p-576" id="p-576" id="p-576" id="p-576" id="p-576" id="p-576" id="p-576"
[576] In certa embodimein nts of the Shiga toxin effecto polypeptidesr of the present invention, one or more amino acid residues may be mutated, inserte ord, deleted in order to increas thee enzymat activic ityof the Shiga toxin effector polypeptide. In certa embodimin ents of the Shiga toxin effector polypepti desof the present invention, one or more amino acid residues may be mutated or deleted in order to reduce or eliminate catalyti and/c or cytotoxic activity of the Shiga toxin effector polypeptide. For example, the catalytic and/o cytotoxicr activ ityof the A Subunits of members of the Shiga toxin family may' be diminished or eliminate by d mutation or truncation. 577. 577. 577. id="p-577" id="p-577" id="p-577" id="p-577" id="p-577" id="p-577" id="p-577" id="p-577" id="p-577" id="p-577"
[577] The cytotoxic ofity the A Subunit sof members of the Shiga, toxin family' may be altere reduced,d, or eliminate byd mutation and/or truncat. ionThe positions labeled tyrosine-77, glutamate-167, arginine-1 tyrosi70, ne-114, and tryptophan-203 have been shown to be important for the catalyt actiic vity of Stx, Stxl, and Stx2 (Hovde C et al., Proc Natl. Acad Sci USA 85: 2568-72 (1988); Deresiewicz R et al., Biochemistry 31: 3272-80 (1992); Deresiewicz R et al., Mol Gen Genet 241: 467-73 (1993); Ohmura M et al., Microb Pathog 15: 169-76 (1993); Cao C et al., Microbiol Immunol 38: 441-7 (1994); Suhan M, Hovde C, Infect Immun 66: 5252-9 (1998)).
Mutating both glutamate-167 and arginine-170 eliminate thed enzymatic activity of Slt- AlI in a cell-fre ribosomee inactiva tionassay (LaPointe P et al., J Biol Chem 280: 23310-18 (2005)). In another approa usingch de novo express ionof Slt-I Al in the endoplasm reticulum,ic mutati ngboth glutamate- and167 arginine-1 70 eliminat Slt-Ied Al fragment cytotoxici at thatty express ionleve (LaPointel P et al., J Biol Chem 280: 23310-18 (2005)). A truncati analon ysis demonstr atedthat a fragment of StxA from residues 75 to 268 still retains significa enzymatnt activityic in vitro (Haddad J et al., J Bacterial 175: 4970-8 (1993)). A truncat fragmed ent of Slt-I Al containi resing dues 1-239 display signifed icant enzymatic activity in vitro and cytotoxicity by de novo express ionin tire cytosol (LaPointe P et al., J Biol Chem 280: 23310-18 (2005)). Expression of a Slt-I Al fragment truncat toed residues 1- 239 in the endoplasm reticic ulum was not cytotoxic because it could not retro translo tocat thee cytosol (LaPointe P et al., J Biol Chern 280: 23310-18 (2005)). 578. 578. 578. id="p-578" id="p-578" id="p-578" id="p-578" id="p-578" id="p-578" id="p-578" id="p-578" id="p-578" id="p-578"
[578] The most critical residue fors enzymat activiic ty'and/or cytotoxici in thety Shiga toxin A Subunit swere mapped to the following residue-positions: asparagin e- 75, tyrosine-77, tyrosi114,ne- glutamate- 167,arginine-1 70,arginine-176, and WO 2016/196344 PCT/US2016/034778 214 tryptophan- among203 others (Di R et al., Toxicon 57; 525-39 (2011)). In particular, a double-mutant construct of Stx2A containing glutamate-E167-to-lysi and ne arginine-176-to-lysi mutatne ions was complete inactivated;ly wherea manys, singl e mutations in Stxl and Stx2 showed a 10-fol reductiond in cytotoxicity. Furthe r, truncation of StxlA to 1-239 or 1-240 reduced its cytotoxici andty, similarly , truncati ofon Stx2A to a conserve hydropd hobic residue reduced its cytotoxic ity.
The most critical residues for bindin eukaryotig ribosc omes and/or eukaryoti c ribosome inhibition in the Shiga toxin A Subunit have been mapped to die following residue-positio arginsnine-172, arginine-176, arginine-1 79,arginine-188, tyrosine- 189, valine-1 91,and leucine-233 among others (McCluskey A et al., PLoS One 7: e31191 (2012). However, certa modifiin cati mayon increase a. Shiga toxin functional activit exhiy bite byd a Shiga toxin effect polypeptideor of die present invention. For example, mutating residue-posit alanmeion -231 in StxlA to glutama incrte eas StxlAed ’s enzymati actc ivit in yvitro (Suhan M, Hovde C, Infect Immun 66; 5252-9 (1998)). 579. 579. 579. id="p-579" id="p-579" id="p-579" id="p-579" id="p-579" id="p-579" id="p-579" id="p-579" id="p-579" id="p-579"
[579] In certain embodimen ofts Shiga toxin effector polypepti desof the prese nt invention derived from SLT-1A (SEQ ID NO:1) or StxA (SEQ ID NO:2), the one or more amino acid residues mutated include substitution of the asparagine at position 75, tyrosi atne position 77, tyrosine at position 114, glutama atte position 167, arginine at position 170, arginine at position 176, and/or substitution of the tryptophan at position 203. Example ofs such substituti onswill be known to the skilled worker base don the prior art, such as asparagine at position 75 to alanine, tyrosine at position 77 to serine, substitution of the tyrosine at position 114 to serine, substitution of the glutama positionte 167 to glutamat subste, ituti ofon tire arginine at position 170 to alanine subs, titution of the arginine at position 176 to lysine , substitution of the tryptophan at position 203 to alanine and/or, substituti ofon the alanine at 231 with glutamat Othere. mutations whic heithe enhancr ore reduce Shiga toxin enzym atic activity and/o cytotoxir city are within the scope of the invention and may be determined using well known techniques and assays disclosed herein. 580. 580. 580. id="p-580" id="p-580" id="p-580" id="p-580" id="p-580" id="p-580" id="p-580" id="p-580" id="p-580" id="p-580"
[580] lire Shiga toxin effector polypepti desand cell-targeti molecng ules of the present invention may optionall be conjugatedy to one or more additional agents, which may include therapeuti agecnts, diagnos agetic nts, and/o other additionalr exogenou mats eri alsknown m the art, including such agents as describe hereid n.In WO 2016/196344 PCT/US2016/034778 215 certain embodiments, the Shiga toxin effecto polypeptider or cell-targetin moleculeg of the prese ntinvention is PEGylated or albuminated, such as, e.g., to provide de- immunization, disrupt furin-cleavage by masking the extende loopd and/or the furin- cleava motifge at the carboxy-terminus of a Shiga, toxin Al fragme derint ved region, improve pharmacokinetic propert iesand/o, improver immunogenicity (see e.g., Wang Q et al., Cancer Res 53: 4588-94 (1993); Tsutsumi Y et al., Proc Natl Acad Set USA 97: 8548-53 (2000); Buse J, El-Aneed X, Nanomed 5; 12372010) 60־); Lim S et al., J Control Release 207-93 (2015)).
V. Gener alFunctions of the Cell-Target Molecing ules of the Present Invention 581. 581. 581. id="p-581" id="p-581" id="p-581" id="p-581" id="p-581" id="p-581" id="p-581" id="p-581" id="p-581" id="p-581"
[581] The functiona asslociatio of Shiga,n toxin effector polypepti desof the present invention with cell-targeti bindinng gregio nsenables the creation of cell-targetin g molecules which selective kill,ly inhibit tire growth of, deliv erexogenou mates rial to, and/o deter ctspecific cell types The. propert ofies the Shiga toxin effector polypeptide of the present invention enabl thee creation of cell-targetin molecg ules wit himprove therd apeu windowstic in chordat ases compar toed prior Shiga toxin effector polypeptides. 582. 582. 582. id="p-582" id="p-582" id="p-582" id="p-582" id="p-582" id="p-582" id="p-582" id="p-582" id="p-582" id="p-582"
[582] For certain embodiments, the cell-target moleingcule of the prese ntinvention provides, afte adminisr tration to a chorda onete, or more of the following: 1) potent and selective killing of target cellsed e.g.,, infected or maligna cellsnt at, low administration dose s,2) linkage stabilit betwey en the cell-target bindinging region and the Shiga toxin effector polypeptide region while the cell-target moleingcule is present in extracellular spaces, 3) low levels of off-target cell deaths and/or unwanted tissue damage, and 4) cell-targete deliveryd of heterologous, CD8+ T-cell epitopes for presentati byon targ cellset in order to initiate desirable, T-cell mediated, immune respons es,such as, e.g., the recruitment of CD8+ T-cells and tire locali zedrelease of cytokine at as tissue locus. 583. 583. 583. id="p-583" id="p-583" id="p-583" id="p-583" id="p-583" id="p-583" id="p-583" id="p-583" id="p-583" id="p-583"
[583] The Shiga toxin effector polypepti des and cell-targeti molecng ules of the present invention are usefi llin diverse applications involving, e.g., cell-killi cellng; growth inhibition; intracellula cargor, delive ry;biologica inforl matio gathering;n immune respons stimulate ion,and/or remedratio of an health condrtion. The Shiga toxin effector polypepti desof the prese ntinvention are useful as components of vario ustherapeut and/oric diagnosti molec cule suchs, as, e.g. ligand-toxin fusions, immunotoxins and/or, immuno-conjugat lirees. cell-target molecing ules of the WO 2016/196344 PCT/US2016/034778 216 present invention are useful as therapeutic and/or diagnos moletic cule suchs, as, e.g., as cell-targe cytotoxic,ting, therapeuti molec cule cels; l-targe nontoxic,ting, deliver y vehicles; and/o celr l-targeti diagnosng, molectic ules for; examples in application s involving the in vivo targeting of specif iccell types for the diagnosis or treatment of a variet ofy diseases including, cancers, immune disorde andrs, microbial infections. 584. 584. 584. id="p-584" id="p-584" id="p-584" id="p-584" id="p-584" id="p-584" id="p-584" id="p-584" id="p-584" id="p-584"
[584] Depending on the embodiment, a Shiga toxin effecto polypeptider or cell - targeting molecule of the present invention may have or provide one or more of the following characteristics or functionalities (1) de-immunization,: (2) protease - cleava resige stanc (3)e, potent cytotoxic at itycertain concentrat (4)ions, intracellula r delive ofry a cargo consisting of an additional material (e.g. a heterologous T-ce,ll epitope), (4) selective cytotoxi city,(6) low off-targ toxiciet inty multicellula r organisms at certain doses or dosages, (7) delive ofry a heterologous T-ce,ll epitop e to the MHC class I presenta tionpathway of a targe celt l,and/o (8)r stimulation of CD8+ T-cell immune response( s).Certain embodiments of the Shiga toxin effector polypepti desand. cell-target molecing ules of the prese ntinvention are multi - functional becaus thee molecule haves two or more of the characteristics or functionalit desiescribe hered in. Certa furtherin embodiments of the cell-targetin g molecules of the prese ntinvention provide all of the aforementione chardacter istics and functionalit in aies single molecule. 585. 585. 585. id="p-585" id="p-585" id="p-585" id="p-585" id="p-585" id="p-585" id="p-585" id="p-585" id="p-585" id="p-585"
[585] The associating, coupling, and/or linking of a cell-target bindinging region (s) with a Shiga, toxin effector polypeptide( ofs) the prese ntinvention, enables the engineerin of gcell-targeti molecng ules wit hShiga toxin function(s) that can produc lese sadverse effect afters administration at certai dosesn or dosages to a multicellul orgaar nis suchm as a mammal. Non-limitin examg ples of advers effe ect s include off-target toxicitie untargets, cytotoed xicit and/oies, unwantedr immune responses Certa. embodin imen ofts the Shiga toxin effecto polypeptidesr and ceil- targeting molecules of the present invention are particular usefully in applicati ons involving administration of a Shiga toxin effecto polypeptr ideand/or cell-targeting molecule to a chordate because of functional propert iessuch, as, e.g., de- immunization, reduced off-target toxicities and/or, targe tedstimulat ofion desirable immune response suchs as via cell-surfa presentace tionof a cell-target moleculeing delivered, CD8+ T-cell epitope. 586. 586. 586. id="p-586" id="p-586" id="p-586" id="p-586" id="p-586" id="p-586" id="p-586" id="p-586" id="p-586" id="p-586"
[586] In certa embodimein nts, the cell-targetin moleculesg of the prese ntinvention are capable of bindin extrg acel lulartarge biomolecult assoces iated with the cel lWO 2016/196344 PCT/US2016/034778 217 surfa ceof particular cel typesl and entering those cells Once. internalized within a targe tedcell type, certain embodiments of the cell-target moleculesing of the invention are capable of routing an enzymatically active cytot, oxic, Shiga toxin effector polypeptide fragment into the cytosol of the targe cellt and eventually killing die cel l.Alternative nontoxily, orc reduced-toxicity variants of die cell- targeting molecules of the prese ntinvention may be used to deliv eradditional exogenou mats eri alsinto targ cellset such, as epitope peptides, proteis, ns, polynucleot andides, detection promoti agents.ng This system is modular, in that any number of diverse bindin gregions can be used to targe a Shigat toxin effector polypeptide of the present invention to various, diverse cell types.
A. De-Immunization for Applications Involving Administration to a Chordate 587. 587. 587. id="p-587" id="p-587" id="p-587" id="p-587" id="p-587" id="p-587" id="p-587" id="p-587" id="p-587" id="p-587"
[587] The de-immunization of the Shiga toxin effector polypepti desof the present invention is accomplished by engineerin disruptg ions of one or more, endogenou s, B-cell and/o CD4+r T-cell epitopes regions of a Shiga toxin A Subunit or Shiga toxin effector polypepti includingde, via mutation and/o truncationr or via the conjugation of a covalently-linke chemidcal structure Beca. use B-cell epitopes often coincide or overlap with epitopes of matur CD4+e T-cells, the disruption of an endogenous, B-cell epitope region ofte simultaneouslyn disrupts an endogenous, CD4+ T-cell epitope or vice versa. 588. 588. 588. id="p-588" id="p-588" id="p-588" id="p-588" id="p-588" id="p-588" id="p-588" id="p-588" id="p-588" id="p-588"
[588] Certain embodimen ofts the Shiga toxin effector polypeptides and cell- targeting molecules of the prese ntinvention are de-immunize withd respect to one or more B-cell and/o CD4+r T-cell epitopes meaning that thes moleculee exhis bit reduced antigenic and/o immunor geni potentic asal compared to prior, Shiga toxin effector polypeptides and cell-targeti moleculeng lackings identical disruptions to the same B-cell and/or CD4+ T-cell epitope or epitope regions and/or lacking any disruption to the same B-cell and/o CD4+r T-cell epitope(s or) epitope region(s . ).
Certa furthein embodimr ents exhibi potentt if not wild-type levels of Shiga toxin A Subunit catalytic domai dependentn cytotoxicity despite the presence of multiple mutations providing the de-immunized property. The de-immunize d,Shiga toxin effector polypeptides and cell-targeti molecng ules of the present invention are useful for applicati onsinvolving the parentera adminisl trati of aon Shiga toxin effector polypeptide and/o cellr -targetin molegcule to a chordate such as, e.g., a mammal, WO 2016/196344 PCT/US2016/034778 218 amphibian, bird, fish reptile, ors, shar k,becaus ofe the reduced likelihood of produci ngundesira bleimmune response invokeds by the administrate molecd ule. 589. 589. 589. id="p-589" id="p-589" id="p-589" id="p-589" id="p-589" id="p-589" id="p-589" id="p-589" id="p-589" id="p-589"
[589] The various de-immunize Shigad, toxin effecto polypeptidesr of the present invention might differ in their antigeni citprofiley whens administer to edvarious chordate species, but all the de-immunized polypeptides of the invention exhibit reduced antigeni cityand/o immunor genicit in aty leas onet organis asm measure byd at leas onet quantitat assiveay. In particular certa, embodimein nts of the cell- targeti moleculesng of the prese ntinvention are de-immunized with respect to a mammalian recipie nt,such as, e.g., the molecul invokese lower quantitie and/s or frequenc ofies "anti -cell-targe moleculeting " antibodies when administere to thatd mamma asl compar toed a refere ncemolecul (e.g.e a related cell-targeti molengcule comprisin a gwild-type Shiga toxin Al fragment). In addition, Shiga toxin effector polypepti desof the prese ntinvention having disruptions of multipl endogee, nous, epitope regions are expected to more greatly reduc edthe probability of the occurre nceof undesirable immune response in sa. chordate recipient of such a polypeptide. 590. 590. 590. id="p-590" id="p-590" id="p-590" id="p-590" id="p-590" id="p-590" id="p-590" id="p-590" id="p-590" id="p-590"
[590] For certa embodin imen ofts the Shiga toxin effecto polypeptidesr and cell- targeting molecules of the prese ntinvention, the de-immunization property(ies is a ) result of the structu change(s)ral which include the disrupt edfurin-cleavage motif at the carboxy-terminus of a Shiga toxin Al fragment derived region. 591. 591. 591. id="p-591" id="p-591" id="p-591" id="p-591" id="p-591" id="p-591" id="p-591" id="p-591" id="p-591" id="p-591"
[591] For certain embodimen ofts the Shiga toxin effecto polypeptidesr and cell- targeting molecules of the prese ntinvention, the de-immunizati properton y(ies is a ) result of the structural change(s which) include the embedding and/o insertr ing of a T-cell epitope which disrupts an endogenou B-cels, and/orl CD4+ T-cell epitope region. 592. 592. 592. id="p-592" id="p-592" id="p-592" id="p-592" id="p-592" id="p-592" id="p-592" id="p-592" id="p-592" id="p-592"
[592] For certa embodimein nts, the desired biological function( ofs) the parental, Shiga toxin polypeptide from which the de-immunized, Shiga toxin effector polypept idewas derived are preserved, such as, e.g., the Shiga toxin A Subuni t functions of promoti cellng ular internalizat direion,ctin intrag cellul routaring, and potent cytotoxicity. Preserva tionrefers to the retention of a minimal leve ofl activity as described herein.WO 2016/196344 PCT/US2016/034778 219 B. Reduced Protease-Cleavage Sensitivity 593. 593. 593. id="p-593" id="p-593" id="p-593" id="p-593" id="p-593" id="p-593" id="p-593" id="p-593" id="p-593" id="p-593"
[593] Certain embodimen ofts the Shiga toxin effector polypeptides and cell- targeting molecules of the prese ntinvention exhibit redu ced protease-cle avage sensitivity as compared to relate moleculesd comprisin wild-g type, Shiga toxin A1 fragment regions Certa. furthein embodimr ents exhibit potent if not optimal, Shiga toxin A Subuni tcatalytic domain dependent cytotoxic despiity te this reduced protease-cle avagesensitivity' and lack of a canonical furin-cleavage event within an intoxicat cell.ed 594. 594. 594. id="p-594" id="p-594" id="p-594" id="p-594" id="p-594" id="p-594" id="p-594" id="p-594" id="p-594" id="p-594"
[594] Certain embodiments of the protease-clea resistant,vage cell-targeting molecules of the prese ntinvention (/.e. a cell-target moleingcule comprisin a gShiga toxin effector polypeptide comprising a disrupted furin-cleavage motif at the carboxy-termi ofnus its Shiga toxin Al fragment region) exhibit improved in vivo tolerability as compared to related molecules comprisi nga wild-type, Shiga toxin Al fragment region. Certa furthein embodimr ents exhibit potent if not optimal, Shiga toxin A Subunit catalyt domaiic dependentn cytotoxic desitypite this reduce d protease-cle avagesensitivity and lac ofk a canonical furin-cleavage event within an intoxicat cell.ed 595. 595. 595. id="p-595" id="p-595" id="p-595" id="p-595" id="p-595" id="p-595" id="p-595" id="p-595" id="p-595" id="p-595"
[595] Previously, it was believed that cytotoxic, Shiga toxin A Subunit constructs comprising Shiga toxin Al fragment catalyti regionsc mus tmainta orin somehow compensate for the naturally occurring proteolytic process ingby furi nwithin intoxicat celed lsin order to preser theve Shiga toxin’s natur adaptaal tions for efficient and potent cytotoxicity. It was unexpecte7 dlydiscovere thatd the furi n cleava eventge was not require ford potent cytotoxic becauseity potent Shiga toxin cytotoxic׳ atity the leve ofl a wild-type Shiga toxin control construct was achieved in the absence of any furin cleavage event at the carboxy-termi ofnus the Shiga toxin A1 fragment despite the presence of a carboxy-ter minamoietyl (see Examples in/ra, and WO 2015/191764). The lack of a furm-cleav eventage withi then intoxicated cell may prevent the efficie liberatnt ionof a Shiga toxin Al fragment- regiolike n and, thus, result in the continued linkage of a relative largely moiety (e.g. great er than 28 kDa in size) to the Shiga toxin Al fragme regint on. However despite this possibilit7, potent,y Shiga toxin cytotoxi wascit yachieved with furm-cleavage deficient constructs comprisi nga Shiga toxin effecto polypeptider regio andn lacking any known compensator featury e(s) such, as, e.g., providing intracellula rWO 2016/196344 PCT/US2016/034778 220 cleava proxge imal to the carboxy-terminus of a Shiga toxin Al fragment derived region (see Examples infra;, WO 2015/191764). 596. 596. 596. id="p-596" id="p-596" id="p-596" id="p-596" id="p-596" id="p-596" id="p-596" id="p-596" id="p-596" id="p-596"
[596] This suggests that the persisten and/orce inefficient release of a relative ly large, molecular moiet linkedy to the A l fragment regio didn not necessar reduceily the potency of Shiga toxin cytotoxicity. Tliis was surprising becaus thee optimal Shiga toxin intoxica tionproce sswas thought to requi reliberati ofon the Shiga toxin Al fragments from all othe larger molecular moieties to efficiently retrotrans locate liberated Al fragments from the endoplasmic reticulum to the cytosol where the Al fragments can form an enzymatically active structure that catalytica inactivateslly the intoxica cellted’s ribosomes In. particular the persis, tence and/o ineffir cient release of a relative largely molecular moiet coveringy the carboxy-terminus of the Shiga toxin Al fragment was expected to interfe withre the Shiga toxin Al fragme’snt natura mechanl ism of efficientl gainingy access to the cytos ol,which involve thes exposure of the Al fragment’s, hydrophobic, carboxy-termina domainl and recognitio of thisn domain by the ERAD system (see Di R et al., Toxicon 57: 525-39 (2011); Li S et ah, PLoS One 7: e41119 (2012)). 597. 597. 597. id="p-597" id="p-597" id="p-597" id="p-597" id="p-597" id="p-597" id="p-597" id="p-597" id="p-597" id="p-597"
[597] The lack of an intoxicated-cell-media furin-ted,cleavage event for a molecule comprising a Shiga toxin A Subunit derivative may be hypothetically compensa ted for. Non-limit ingexamples of potentia compensl, ator approachey includes 1) terminatin oneg carboxy-terminus of the construc witht the carboxy-terminus of a Shiga toxin Al fragment- polypeptidelike region, 2) producing the Shiga toxin derived constr suchuct that the Shiga toxin A Subunit polypeptide is already nicked near the carboxy-terminus of its Shiga toxin Al fragment- polyplike epti de,3) engineerin a heteg rologous and/or ectopic protease site that can functionally substitute for the lack of the native Shiga, toxin, furin-cleava event,ge and 4) a combinat ionof approac 3 hand 4. 598. 598. 598. id="p-598" id="p-598" id="p-598" id="p-598" id="p-598" id="p-598" id="p-598" id="p-598" id="p-598" id="p-598"
[598] In the first approa ch,the carboxy-terminus of the Shiga toxin Al fragme nt- like polypeptide is not covere byd any carboxy-termina moiety',l and, thus the, carboxy-termi ofnus the Shiga toxin Al fragment- polypeptidelike is permanently expose ford recognit by'ion the ERAD machine ry'in the endoplasmic reticulum. In the las threet approaches the ,Shiga toxin Al fragment- polypelike ptide can be designed to intracellularly' dissocia frteom one or more othe componer ntsof the construct by the time the molecule reache thes endoplasmic reticulum of an intoxicat ceedll such that in the endoplasm reticiculum the carboxy-terminus of the WO 2016/196344 PCT/US2016/034778 221 Shiga toxin Al fragment- polypeptidelike become expos se ford recognition by the ERAD machine ry.For example, a cytotoxic molecule comprisi nga Shiga toxin A Subunit effecto polypeptider coul bed pretrea withted a protea tose nick the polypeptide region near the carboxy terminus of the A1 fragment- regionlike prior to contacting a target cell Alter. natively, the cytotoxic molecule could be engineered to comprise a protease sit ewhich is cleaved by an intracellula protear ofse the target cell. 599. 599. 599. id="p-599" id="p-599" id="p-599" id="p-599" id="p-599" id="p-599" id="p-599" id="p-599" id="p-599" id="p-599"
[599] These hypothetical approac hesfor designing Shiga toxin A Subuni teffector polypepti deswhich compensa forte the lack of an intoxicated-cell-mediated furin- , cleava eventge may significant altly erthe efficiency and potency of cytotoxicity as compared to a wild-type Shiga holotoxin, or Shiga, toxin A Subunit construc t comprising only wild-type sequence whichs include the optimal, naturally occurring, furin-cleavage site. For example, currently no compensat approaory chrelying on a targe celt endopl rote otherase than furin is known which can provide fully compensatory cytotoxic equivaity lent to furin cleavage and alternat celivelular proteas toes furin like calpains have been shown to be les sefficient in facilitating Shiga toxin cytotoxici (Gatyrre Od et al., Exp Cell Res 218: 39-49 (1995); Garred O et al., J Biol Chem 270: 10817-21 (1995); Kurmanova A et al., Biochem Biophys Res Commun 357: 144-9 (2007)). 600. 600. 600. id="p-600" id="p-600" id="p-600" id="p-600" id="p-600" id="p-600" id="p-600" id="p-600" id="p-600" id="p-600"
[600] The present invention provides furm-cleav resiagestant Shiga toxin A Subunit effecto polypeptidesr which are potently cytotoxic, whether due to compensation for a lac ofk a furin cleavage event withi then intoxicat celled or due to some unexplained reason. Certain cell-targe ting molecules of the presen t invention are at leas ast efficientl andy potently cytotoxic as cell-target molecing ules comprising protease-cle avagesensitive, wild-type Shiga toxin effector polypeptide regions (see Examples, infra).
C. Improved Stabili tyand Vivo Tolerability' 601. 601. 601. id="p-601" id="p-601" id="p-601" id="p-601" id="p-601" id="p-601" id="p-601" id="p-601" id="p-601" id="p-601"
[601] In certa embodimein nts, the molecules of the prese ntinvention (e.g. cell - targeti moleculesng of the invention) exhibi increat sed stabilit and/oy imprr oved in vivo tolerability as compared to more furin-cleavage sensitive analogs and/or less de-immunized analogs (an analog being a close relaly ted molecule lacking one or more structural features of the prese ntinvention).[602] The increased stability of a cell-target molingecule compare tod a reference molecule can be exhibited in vitro and/or in vivo. The stabil itofy a therape uticor diagnost molic ecule over time is an important feature and can affect for which applications the molecule may be practic allyemployed. Molecular stabilit incly udes in vitro and in vivo, such as, e.g., stabilit wity hin an organi smafter administration and during storage over a range of temperat uresand concentrati Forons. certain immunotoxins or ligand-toxin fusions the, stabil itofy the linka gebetween the toxi andn other compone ntscan affect the amount of non-specific toxicit causedy by the presence and/or quant ityof untargeted toxin over tim ewithi then organism. 603. 603. 603. id="p-603" id="p-603" id="p-603" id="p-603" id="p-603" id="p-603" id="p-603" id="p-603" id="p-603" id="p-603"
[603] Certain cell-target molecing ules of the present invention exhibit reduce nond ­ specific toxici inty vivo, manifest ased increased in vivo tolerab ilias comparety tod more protease-cleavage sensitive variants In .vivo tolerabil canit bey determined by the skilled worker using techniq uesknown in the art and/or described herei n.In additi onto assessing in vivo tolerabil usingity mortal signity, sof morbidit may y be used for assessing in vivo tolerabili suchty, as, e.g., aspec tsof body weight, physical appearance, measurabl clinie cal signs, unprovoked behavior and, responses to external stimu li(see e.g. Morton D, Griffiths P, Vet Rec 116: 431-43 (1985); Montgomery C, Cancer Bull 42: 230-7 (1990); Ullman-Cul lereM, Folt C,z LabAnim Sci 49: 319-23 (1999); Clingerman K, Summers L, J Ain Assoc Lab Anim Sci 51: 31 -6 (2012)). Euthanasia may be used in respons toe sign ofs morbidity and/or moribundity and, thus, creat a e mortali timtye-poin Fort. example, a decrease in body weight of 15-20% in 2-3 days can be used as a sign of morbidi tyin rodent ands as a justificati foron euthanization (see e.g. Institute of Laboratory Animal Research 2011. Guide for the care and use of laboratory animals, Sth ed., Washington, DC, U.S.: National Academies Press). 604. 604. 604. id="p-604" id="p-604" id="p-604" id="p-604" id="p-604" id="p-604" id="p-604" id="p-604" id="p-604" id="p-604"
[604] The improved in vivo tolerab iliobservedty for exemplary, cell-targe ting molecul ofes the present inventi onas compare tod more furin-cleava sensige ti veanalogs sugges thatts much higher doses of these cell-target moleingcul ofes the invention may be safel admiy niste tored mammals as compared to the doses of relat moled ecul es comprising a furin-cleava sensige tive, Shiga toxi effen ctor polypepti region.de Certain cell-target moleculing ofes the inventi onmight exhibit reduce non-specificd toxicity as compared to more protea sensise ti vevarian becats use the protea resisse tance serves to protect and preserve the linkage between the Shiga toxi effectn compoor nent and the cell-target moieingty component. -222-WO 2016/196344 PCT/US2016/034778 223 605. 605. 605. id="p-605" id="p-605" id="p-605" id="p-605" id="p-605" id="p-605" id="p-605" id="p-605" id="p-605" id="p-605"
[605] In addition, in vivo tolerabili forty cell-targeti moleculeng ofs the present invention may be relate to dtli ede-immunization propert ofies a given cell-targeting molecule. Thus, higher doses of such, de-immunize cell-d, target moleculeing ofs the invention may be safely administer to edmammals as compar toed the doses of related molecules comprisin ang "un-de-immunize" ord less de-immunize Shigad, toxin effector polypeptide (e.g. a wild-type Shiga toxin Al fragment). 606. 606. 606. id="p-606" id="p-606" id="p-606" id="p-606" id="p-606" id="p-606" id="p-606" id="p-606" id="p-606" id="p-606"
[606] In addition, certai moleculen ofs the invention exhibit increas halfed -live s, both in vitro and/or in vivo, as compared to more protease-cle avagesensitive variants Molecula. stabilityr can be assaye byd determini theng half-l ofife a molecule of inter estwit hregard to the associat ofion its components. Certa in embodiments of the molecules of the invention will have longe halfr -li vesas compared to furin-cleavage sensitive variants esp,eciall withy rega rdto the continued associati ofon the Shiga toxin effector polypept idecomponen andt one or more othe comr ponents. For example, certai embodimn ents of the molecules of the invention will have longe half-livesr with regard to the continued association of the Shiga toxin effector polypeptide component and another component, e.g. a cell- targeting binding region as, compared to a furin-cleava segensitive variant wherein the furin-cleavage sensitive site(s lie) sbetween those two components.
D. Cell-Kill via Shiga Toxin A Subuni tCytotoxicity 607. 607. 607. id="p-607" id="p-607" id="p-607" id="p-607" id="p-607" id="p-607" id="p-607" id="p-607" id="p-607" id="p-607"
[607] Certain embodimen ofts the Shiga toxin effector polypeptides and cell- targeting molecules of the prese ntinvention are cytotoxic. Certa infurthe r embodiments of the cell-target molecing ules of the prese ntinvention are cytotoxic only due to the presence of one or more Shiga toxin effector polypept ide components. The A Subunit sof member ofs the Shiga toxin famil eachy comprise an enzymatically active polypeptide regio capn able of killing a eukaryot ceicil once m the cell’s cytosol. Because members of the Shiga toxin family are adapted to killing eukaryotic cells, molecule derives fromd Shiga toxins, such as, e.g., molecules comprising certai embodimen nts of the Shiga toxin effector polypepti des of the prese ntinvention can exhibit potent cell-kill activities. 608. 608. 608. id="p-608" id="p-608" id="p-608" id="p-608" id="p-608" id="p-608" id="p-608" id="p-608" id="p-608" id="p-608"
[608] For certain embodimen ofts the cell-targetin molecg ules of the present invention, upon contacti a ngcell physically coupled wit han extracel targelular t biomolec uleof the binding regio ofn tlie cell-targeti molengcule (e.g. a targe t positiv cell),e the cell-targetin molecg ule is capable of causing death of the cel l.Forcerta furtherin embodiments, the CDs0 value of the cell-targeti molecng ule is less than 5, 2.5, 1, 0.5, or 0.25 nM, which is vastl morey poten thant an untarget wiled,­d type, Shiga toxi effen ctor polypept ide(e.g. SEQ ID NO:4). 609. 609. 609. id="p-609" id="p-609" id="p-609" id="p-609" id="p-609" id="p-609" id="p-609" id="p-609" id="p-609" id="p-609"
[609] Cell-kil mayl be accomplish usinged a molecule of the present inventi on under varie conditd ions of target cell suchs, as, e.g., an ex vivo manipulated target cell, a target cell cultured in vitro, a target cel witl hin a tissue sampl culte ured in vitro, or a target cell in an in vivo setti nglike within a multicellul organism.ar 610. 610. 610. id="p-610" id="p-610" id="p-610" id="p-610" id="p-610" id="p-610" id="p-610" id="p-610" id="p-610" id="p-610"
[610] In certai embodimn ents, the Shiga toxi effectn polypeor ptide ands cell­ target moleing cules of the present inventi comprion se(1) a de-immunized, Shiga toxin effector sub-region, (2) a protease-cleavage resistant region near the carboxy­ terminus of a Shiga toxi Aln fragment derived region, (3) a carboxy-termi nal, endoplasm retiic culum retention/retrie signvalal moti f;and/or (4) a heterologous, T- cel epitopel embedded or insert region;ed however, for certain further embodiments, these structural modificatio dons not significant alterly the potency of Shiga toxi n cytotoxi ascity compared to a reference molecul compries sing a wild-type Shiga toxin A Subunit polypepti suchde, as, e.g., a wild-type Shiga toxi Aln fragment .
Thus, Shiga toxi effen ctor polypeptides and cell-targeti molengcule ofs the present invention which are de-immunize proteased, cleavage resistant, and/or carrying embedded or inserte heted, rologo epitusopes can maintain potent cytotoxici whilty e providing one or more various other functionali orti propertes ies. 611. 611. 611. id="p-611" id="p-611" id="p-611" id="p-611" id="p-611" id="p-611" id="p-611" id="p-611" id="p-611" id="p-611"
[611] Already cytotoxic cell-target moleingcules comprising Shiga toxin effect or polypeptide mays be engineered by the skilled worker using the informati andon metho dsprovid edherei ton be more cytotoxi and/orc to have redundant, backup cytotoxici operaties tin viag completel differey mechnt anism Theses. multiple cytotoxic mechanisms may complement eac hother by their diversi ofty functions (such as by providi ngpotent killin viag two mechanisms of cell-killi directng, and indirect, as well as mechanisms of immuno-stimula totion the loca area),l redundantly backup each other (such as by providi ngone cell-kill mechanising inm the absenc ofe the other mechanisms—like if a target cell is resistant to or acquires some immunit toy a subset of previously active mechanisms), and/or protec agait nst developed resistance (by limiting resistance to the les probabls site uation of the malignant or infect celled blocking multiple, differe cell-knt illi mechanng isms simultaneously). -224-WO 2016/196344 PCT/US2016/034778 225 E. Deliven7 of a T-Cell Epitope for MHC Class I Presentation on a Cell Surface 612. 612. 612. id="p-612" id="p-612" id="p-612" id="p-612" id="p-612" id="p-612" id="p-612" id="p-612" id="p-612" id="p-612"
[612] In certa embodimein nts, the Shiga toxin effector polypepti desand cell- targeting molecules of the prese ntinvention comprise a T-cell epitope, which enables the engineering of "T-cell epitope deliver"ing molecule withs virtua lly unlimited choices of epitope-peptide cargos for delivery and cell-surfa ce presentation by a nucleat chordateed, cell For. certa embodimein nts, the Shiga toxin effector polypeptides and cell-targeti molecng ules of the present invention are each capable of deliver ingone or more T-cell epitope asss, ociate withd the Shiga toxin effector polypeptides and/o cer ll-targeti molengcule tos, the proteasome of a cell .
The delivered T-cell epitope are then proteolyt procesic sed and presented by the MHC cla ssI pathway on the surface of the cell By. engineerin MHCg clas I s epitopes into cell-targeti molengcule thes, targete deliveryd and presentation of immuno-stimulator antigensy may be accomplished in order to harne andss direc a t benefici funcal tion( ofs) a chordate immune syste .m 613. 613. 613. id="p-613" id="p-613" id="p-613" id="p-613" id="p-613" id="p-613" id="p-613" id="p-613" id="p-613" id="p-613"
[613] For certain embodiments, the Shiga, toxin effector polypeptide or cell- targeti moleng cule of the present invention is capable of deliver inga T-cell epitope to a MHC class I molecule of a cell for cell-surfa presce entation. In certain embodiments, the Shiga toxin effector polypeptide or cell-target moleculeing of the present inventi comprion ses a heterologous, T-cell epitope whether, as an additional exogenou materials or embedded or inserted within a Shiga toxin effector polypeptide. For certain furthe embodiments,r the Shiga toxin effecto polypeptr ide or cell-target moleingcule of the prese ntinvention is capabl ofe delivering an embedded or inserted T-cell epitope to a MHC clas I smolecule for cell-surfa ce presentation. 614. 614. 614. id="p-614" id="p-614" id="p-614" id="p-614" id="p-614" id="p-614" id="p-614" id="p-614" id="p-614" id="p-614"
[614] For certain em bodiment dies, Shiga toxin effector polypeptide of the present invention is capable of deliver inga T-cell epitope, which is embedded or inserted in the Shiga toxin effecto polypr epti tode, a MHC class I molecule of a cell in which the Shiga toxin effecto polypeptider is prese ntfor presentat ofion the T-cell epitope by the MHC class I molecule on a surface of the cell For. certa furthein r embodiments, the T-cell epitope is a heterologous T-ce,ll epitope .For certain furthe embodr iment thes, T-cell epitope functions as CD8+ T-cell epitope, whether already known or identif iedin the futur usinge methods which are currently routine to the skilled worker.WO 2016/196344 PCT/US2016/034778 226 615. 615. 615. id="p-615" id="p-615" id="p-615" id="p-615" id="p-615" id="p-615" id="p-615" id="p-615" id="p-615" id="p-615"
[615] For certain embodiments, the cell-target moleingcule of the present invention is capable of deliver inga T-cell epitope, which is associated wit hthe cell-targeting molecule to ,a MHC class I molecule of a cell for presentat ofion the T-cell epitope by the MHC class I molecule on a surface of the cell. For certain furthe r embodiments, the T-cell epitope is a heterologous T-ce,ll epitope which is embedde d or inserted in the Shiga toxin effector polypeptide For. certain further embodiments, the T-cell epitope functions as CD8+ T-cell epitope, whether already known or identifie ind the futur usinge methods which are currently routine to the skill ed worker. 616. 616. 616. id="p-616" id="p-616" id="p-616" id="p-616" id="p-616" id="p-616" id="p-616" id="p-616" id="p-616" id="p-616"
[616] For certa embodimein nts, upon contact aing cell with the cell-targetin g molecule of the prese ntinvention, the cell-targeti molecng ule is capable of delivering a T-cell epitope-peptide, which is associated with the cell-targetin g molecule to ,a MHC class I molecule of tire cell for presentation of the T-cell epitope-pept byide the MHC clas I smolecule on a surface of the cell For. certa in further embodiment thes, T-cell epitope-pept iside a heterologous epitope which is embedded or inserted in a Shiga toxin effector polypeptide For. certain further embodiments, the T-cell epitope-pepti functide ons as CD8+ T-cell epitope, whether already known or identif iedin the futur usinge methods which are currently routine to the skilled worker. 617. 617. 617. id="p-617" id="p-617" id="p-617" id="p-617" id="p-617" id="p-617" id="p-617" id="p-617" id="p-617" id="p-617"
[617] The addition of a heterologous epitope into or presence of a heterologous epitope in a cell-target moleingcule of the present inventi on, whether as an additional exogenou mates rial or embedded or insert withied a.n Shiga toxin effector polypepti enablesde, methods of using such cell-target moleculeing fors the cell - targe teddeliver ofy a chosen epitope for cell-surfa presce entati byon a nucleate d, targe cellt within a chordate. 618. 618. 618. id="p-618" id="p-618" id="p-618" id="p-618" id="p-618" id="p-618" id="p-618" id="p-618" id="p-618" id="p-618"
[618] One function of certain, CD8+ T-cell hyper-immunized, Shiga toxin effector polypepti desand cell-target molecing ules of the prese ntinvention is tire deliver ofy one or more T-cel epitope-l peptides to a MHC clas I smolecule for MHC clas I s presentation by a cell Deli. veiy' of exogenous, T-cell epitope-peptides to the MHC class I system of a target cell can be used to induc thee targe cellt to prese ntthe T- cell epitope-pept mide associatio withn MHC class I molecules on the cell surface, which subsequently lead tos the activat ofion CD8+ effecto T-cellsr to atta theck targe cell.t[619] The skilled worker, using technique knowns in the art, can associate, couple, and/or link certai Shigan, toxin effector polypepti desof the present invention to various other cell-targeti bindingng region to create cell-target moleingcule ofs the present invention which target specific, extracel lultargetar, biomolecul physicales ly coupled to cell ands promote target-cel interl naliz atiof theseon cell-target ing molecule Als. lnucleated vertebrat celle ares believed to be capable of present ing intracellul epitaropes using the MHC clas I ssystem Thus,. extracell targetular biomolecul ofes the cell-targeti molengcules of the invention may in principle target any nucleated vertebrat cel eforl T-cell epitope delivery to a MHC cla ssI presentat pathwaion ofy such a cel l. . 620. 620. 620. id="p-620" id="p-620" id="p-620" id="p-620" id="p-620" id="p-620" id="p-620" id="p-620" id="p-620" id="p-620"
[620] The epitope-deliveri functing ons of the Shig atoxi effectn polypeptidesor and cell-targeti molengcule ofs the present inventio cann be detected and monitored by a variet ofy standa methord dsknown in the art to the skille workerd and/or described herei n.For example, the ability of cell-targeti moleculesng of the present inventio n to deliv era T-cel epitope-peptil andde drive presentat ofion the epitope-pepti byde the MHC cla ssI syste ofm target cell mays be investigat usinged various in vitro and in vivo assays incl, uding, e.g., the direct detection/visuali ofzati MHCon clas s I/peptide complexes mea, surem entof binding affiniti fores the heterologous, T-cell epitope-pept toide MHC cla ssI molecules and/or, measureme ofnt functional consequences of MHC cla ssI-peptide compl expresentat onion target cell bys monitori cytotng oxic T-lymphocyt (CTL)e response (sees e.g. Example infrs, a). 621. 621. 621. id="p-621" id="p-621" id="p-621" id="p-621" id="p-621" id="p-621" id="p-621" id="p-621" id="p-621" id="p-621"
[621] Certa assin ays to monitor this functi onof the polypepti desand molecule ofs the present invention involve the dire ctdetecti ofon a specific MHC cla ssI/peptide antigen complex in vitro or ex vivo. Common methods for direct visualizati andon quantitat ofion peptide-MHC clas I scomplexe involves various immuno-detect ion reage ntsknown to the skilled worker. For example, specific monoclonal antibodie s can be developed to recognize a particular MHC/class I/peptide antigen complex.
Similar ly,solubl multie, meric T cel recel ptor suchs, as the TCR-STAR reage nts (Aitor Bioscience Corp., Miramar, FL, U.S.) can be used to direct visually ize or quantit specifiate MHCc I/antigen complexes (Zhu X et al., J Immunol 176: 3223-32 (2006)). These specifi mAc bs or solubl multe, imer T-cellic receptors may be used wit hvarious detecti meton hods, including, e.g. immunohistochem flowistry, cytometry, and enzyme-linked immuno assa (ELISA).y -227-WO 2016/196344 PCT/US2016/034778 228 622. 622. 622. id="p-622" id="p-622" id="p-622" id="p-622" id="p-622" id="p-622" id="p-622" id="p-622" id="p-622" id="p-622"
[622] An alternati methodve for direc identificationt and quantificat ofion MHC I/peptide complexe involvess mass spectrome analysestry such, as, e.g., the ProPresent Antige Presentn ation Assay (Prolmmune, Inc., Sarasota, FL, U.S.) in which peptide-MCH class I complexes are extrac fromted the surface ofs cells, then the peptide ares purified and identifie byd sequenci ngmass spectromet (Falry' kK et al., Nature 351: 290-6 (1991)). 623. 623. 623. id="p-623" id="p-623" id="p-623" id="p-623" id="p-623" id="p-623" id="p-623" id="p-623" id="p-623" id="p-623"
[623] In certa assain ysto monitor the 'T-cell epitope delivery' and. MHC clas I s presentation function of the polypepti desand molecules of the prese ntinvention involve computational and/or experimental methods to monitor MHC cla ssI and peptide binding and. stability. Several software programs are available for use by' the skill edworker for predicting the bindin resg ponse ofs peptid esto MHC class I alleles such, as, e.g., The Immune Epitope Databas ande Analys Resis ource (IEDB) Analys Resis ourc MHC-e I binding prediction Consensus tool (Kim Y et al., Nucleic Acid Res 40: W5 25-30 (2012). Severa experil menta assaysl have been routinely' applie d,such as, e.g., cell surface bindin assag ysand/o surfr ace plasmon resonance assa ysto quanti fyand/o comparer bindin gkinetics (Mile sK et al., Mol Immunol 48: 728-32 (2011)). Additiona othelly, MHr C-peptide binding assa ysbase don a measure of the ability of a peptide to stabili theze ternary MHC-peptide complex for a given MHC class I allele as ,a comparison to known contro havels, been develope d (e.g., MHC-peptide binding assay from Prolmmmune, Inc.). 624. 624. 624. id="p-624" id="p-624" id="p-624" id="p-624" id="p-624" id="p-624" id="p-624" id="p-624" id="p-624" id="p-624"
[624] Alternati vely,measurement of thes consequence of MHC clas I/pes ptide antigen complex presentation on the cell surfac cane be performed by monitorin theg cytotoxic T-cell (CTL) respons toe the specifi complex.c These measurements by include direct labeling of the CTLs with MHC clas Is tetrame or pentr amer reagents.
Tetramers or pentam ersbind directly to T cell recepto ofrs a particular specificity, determined by' the Major Histocompatibility Complex (MHC) allele and peptide complex. Additionall tire y,quantificat ofion releas cytokines,ed such as interferon gamma or interleukins by ELISA or enzyme-linked immunosp ot(ELIspot) is commonly assayed to identify specif icCTL responses The. cytotoxic capacit ofy CTL can be measured using a number of assays, including the classica 51 l Chromium (Cr) release assay or alternat non-raive dioac cytotoxicitytive assa ys(e.g., CytoTox96® non-radioa ctikits veand CellTox™ CellTiter-GLO® kits availa ble from Promega Corp., Madison, WI, U.S.), Granzyme B ELISpot, Caspas Actie vity Assays or LAMP-1 translocation flow cytometr assaic ys. To specifically monitor WO 2016/196344 PCT/US2016/034778 229 tiie killing of targe cellst carboxy, fluoresc diacein eta succite nimidyl ester (CFSE) can be used to easily and quickly labe al cell population of interest for m vitro or in vivo investigatio to monitorn killing of epitope specif icCSFE label edtarge celt ls (Durward M et al., J Vis Exp 45 pii 2250 (2010)). 625. 625. 625. id="p-625" id="p-625" id="p-625" id="p-625" id="p-625" id="p-625" id="p-625" id="p-625" id="p-625" id="p-625"
[625] In vivo response to sMHC clas I spresenta tioncan be followed by administerin a MHCg class I/antige promotin agentng (e.g., a peptide, protein or inactivated/attenuated virus vaccine) follow byed challe ngewith an active agen t (e.g. a virus and) monitorin resgponse to sthat agent, typical inly comparison with unvaccinated controls. Ex vivo samples can be monitored for CTL activity wit h methods similar to those described previousl (e.gy. C TL cytotoxicity assa ysand quantificati of oncytoki releasne e). 626. 626. 626. id="p-626" id="p-626" id="p-626" id="p-626" id="p-626" id="p-626" id="p-626" id="p-626" id="p-626" id="p-626"
[626] HLA-A, HLA-B, and/or HLA-C molecules are isolated from the intoxicated cel lsaft erlysis using immune affinity (e.g., an anti-MHC antibody "pulldown־’ purification) and the associated peptides (i.e., the peptides presente byd the isolate d MHC molecules are) recovere fromd the purified complexes. The recove red peptides are analyzed by sequencing mass spectrometr They. mass spectrome try data is compared against a protei databasen library' consist ingof the sequence of the exogenou (non-ss elf) peptide (T-cell epitope X) and the international protei inden x for humans (representi "selfng’ or non-immunogenic peptides) The. peptides are ranke byd significanc acce ordi tong a probabil itydataba se.All detecte antigenicd (non-se lf)peptide sequences are liste d.The data is verifie byd searching against a scrambled decoy database to reduce fals hitse (see e.g. Ma B, Johnson R, Mol Cell Proteomics 11: 0111.014902(2012)). The results will demonstrat that epeptid es from the T-cell epitope X are presented in MHC complexes on the surfac ofe intoxicat targed celet ls. 627. 627. 627. id="p-627" id="p-627" id="p-627" id="p-627" id="p-627" id="p-627" id="p-627" id="p-627" id="p-627" id="p-627"
[627] The set of presented peptide-antigen-MHC complexe cans vary between cel ls due to the antigen-specifi HLAc molecules expresse T-cellsd. can then recognize specif icpeptide-antigen-MHC complexes display oned a cell surfa ceusing different TCR molecule wits hdiffer entantigen-specificities. 628. 628. 628. id="p-628" id="p-628" id="p-628" id="p-628" id="p-628" id="p-628" id="p-628" id="p-628" id="p-628" id="p-628"
[628] Because multiple T-cell epitopes may be delivered by a cell-targeting molecule of the invention, such as, e.g., by embedding two or more differ entT-cel l epitopes in a single proteasome deliver ingeffector polypepti ade, singl cell-e targeting molecule of the invention may be effective chordate of thes same species with differ entMHC clas vanants,s such as, e.g., in humans wit hdiffer entHLA WO 2016/196344 PCT/US2016/034778 230 alleles Ulis. may allow for the combining within a single molecule of differ entT- cell epitop eswith different effectivenes in diffs erent sub-populations of subjects base don MHC comple proteinx diversi andty polymorphis Forms. example, human MHC complex protei ns,HLA protei ns,vary־ among humans base don geneti c ancestry, e.g. African (sub-Sahara Amen), rindian, Caucasiod, Mongoloid, New Guinean and Australian or Pacifi, islander.c 629. 629. 629. id="p-629" id="p-629" id="p-629" id="p-629" id="p-629" id="p-629" id="p-629" id="p-629" id="p-629" id="p-629"
[629] 'The applications involving the T-cell epitope deliveri polypepting desand molecules of the present invention are vast. Every nuclea tedcel inl a mammalia n organis maym be capable of MHC cla ss1 pathway presentati ofon immunogenic, T- cell epitope-peptides on their cell oute surfar ce compls exed to MHC clas I s molecules. In addition, the sensitivi ofty T-cell epitope recognitio is son exquisite that only a few' MHC-I peptide complexe ares required to be presented to result in an immune response, e.g., even presenta tionof a single comple canx be sufficient for recognitio by nan effector T-cell (Sykulev ¥ et al., Immunity 4: 565-71 (1996)). 630. 630. 630. id="p-630" id="p-630" id="p-630" id="p-630" id="p-630" id="p-630" id="p-630" id="p-630" id="p-630" id="p-630"
[630] The activati ofon T-cell response ares desired characteristics of certa anti-in cancer, anti-neoplas anti-tic,tumor, and/or anti-microbial biologic drugs to stimulate the patien’s town immune system towar targed tedcells. Activatio of na robust and strong T-cell response is als oa desired characterist of manyic vaccine Thes. presentation of a T-cell epitope by a targe cetll within an organi smcan lead to the activati ofon robust immune response to sa targe cetll and/o itsr general loca withile n an organism. Thus, the targe teddelivery־ of a 'T-cell epitope for presenta tionmay be utilize ford as a mechanis form activating T-cell responses during a therapeutic regime. 631. 631. 631. id="p-631" id="p-631" id="p-631" id="p-631" id="p-631" id="p-631" id="p-631" id="p-631" id="p-631" id="p-631"
[631] Hie presentat ofion a T-cell immunogeni epitc ope-pept byide the MHC cla ss I system targets the presenting cell for killing by CTL-mediated lysis and also triggers immune stimulation in the loca micrl oenvironment By engineering. immunogeni epitopec sequences within Shiga toxin effector polypept ide compone ntsof target-cell-intemalizi therapeuting molecc ules, the targe teddelive־ ry and presenta tionof immuno-stimulat antiorygens may be accomplished. The presentation of immuno-stimulat non-selfory antige ns,such as e.g. known viral antigens with high immunogenici byty, target cel lssignal tos othe immuner cells to destroy the targ cellset as wel asl to recruit more immune cells to the area. 632. 632. 632. id="p-632" id="p-632" id="p-632" id="p-632" id="p-632" id="p-632" id="p-632" id="p-632" id="p-632" id="p-632"
[632] The presentat ofion an immunogenic, T-cell epitope-pept byide the MHC class I complex targets the presenting cell for killing by CTL-mediated cytolysis.WO 2016/196344 PCT/US2016/034778 231 The presentati byon targete celd lsof immuno-stimulator non-selfy antigens such, as, e.g., known vira epitope-l peptides with high immunogenici canty, signa tol other immune cells to destroy the targe cellst and recru moreit immune cells to the target cell site within a chordate. 633. 633. 633. id="p-633" id="p-633" id="p-633" id="p-633" id="p-633" id="p-633" id="p-633" id="p-633" id="p-633" id="p-633"
[633] Tims, alrea cytody toxic molecule suchs, as e.g. therapeu ortic potentia lly therape uticmolecules comprisin Shigag toxin effector polypeptide mays, be engineered using methods of the prese ntinvention into more cytotoxic molecule s and/o tor have an additional cytotoxic mechanism operating via delivery of a T-cell epitope presenta, tion, and stimulat ofion effector T-cell s.These multiple cytotoxic mechanisms may complement each othe (sucr h as by providing both direct target- cell-killing and indirec (CTL-t mediated cell-kill) redundantling, backupy each other (such as by providing one mechanism of cell-killi in ngthe absenc ofe the other), and/o proter againstct the developme ofnt therapeut resiic stance (by limiting resistance to the les probables situati onof the malignant or infecte cedll evolving to block two differ entcell-kil lingmechanis mssimultaneously). 634. 634. 634. id="p-634" id="p-634" id="p-634" id="p-634" id="p-634" id="p-634" id="p-634" id="p-634" id="p-634" id="p-634"
[634] In addition, a cytotoxic molecule comprising a Shiga toxin effector polypeptide region that exhibits catalytic-base cytotoxicd mayity be engineer byed the skilled worker using routi nemethods into enzymatically inactive variants For. example, the cytotoxic Shiga toxin effector polypeptide component of a cytotoxic molecule may be conferre withd reduce actd ivity and/o renderer inactid veby tire introduc tionof one or mutations and/o truncar tions such that the resulting molecule can still be cytotoxic via its ability to deliver a T-cell epitope to the MHC cla ssI system of a targ ceetll and subseque ntpresentat toion the surfa ceof the targe cell.t In another exampl ae, T-cell epitope may be inserted or embedded into a Shiga toxin effector polypeptide such that the Shiga toxin effecto polypeptider is inactivate by7 d the added epitope (see e.g. WO 2015/113007). Hiis approach remov esone cytotoxic mechanism while retaini orng adding another and may also provide a molecule capable of exhibiting immuno-stimulat to ionthe local are a,of a targe t cell(s) within an organism via delivered T-cell epitope presentati oron "antige n seedin"g. Furthermo non-cytotoxicre, variants of the cell-target molecing ules of the present invention whic hcomprise embedded or inserte heted, rologous T-ce,ll epitopes may be useful in applications involving immune-stimulation within a chordate and/o labelingr of targ cellset within a chordate wit hMHC class I molecule display epitopeed s.WO 2016/196344 PCT/US2016/034778 232 635. 635. 635. id="p-635" id="p-635" id="p-635" id="p-635" id="p-635" id="p-635" id="p-635" id="p-635" id="p-635" id="p-635"
[635] The abilit toy deliver a T-cell epitope of certa Shigain toxin effector polypepti desand cell-targeti molecng ules of the present invention may be accomplished under varie condid tions and in the presence of non-targe bystanderted cell suchs, as, e.g., an ex vivo manipula tedtarg celet l,a targe cellt cultur ined vitro, a targe cetll within a tissue sample cultured in vitro, or a targe cetll in an in vivo setting like within a multicellul organar ism.
F. Cell-Kill via Targete Cytotoxicityd and/o Engagementr of Cytotoxic T-Cells 636. 636. 636. id="p-636" id="p-636" id="p-636" id="p-636" id="p-636" id="p-636" id="p-636" id="p-636" id="p-636" id="p-636"
[636] For certain embodiments, the cell-target molecing ule of the present invention can provide 1) delivery’ of a T-cell epitope for MHC class I presenta tionby a targe t cell and/or 2) potent cytotoxici *7 * * **1 . Forty certa embodimein nts of the cell-targetin g molecules of the prese ntinvention, upon contact aing cell physically coupled wit h an extracellu targlar biomoleculet ofe the cell-target bindinging region, tire cell - targeting molecule of the invention is capabl ofe causing death of the cell The. mechanism of cell-kill may be direct, e.g. via the enzy7mati activityc of a toxin effector polypeptide region, or indirec viat CTL-mediated cytolysis. 1. Indirec Cell-Killt via. T-Cell Epitope Delivery' and MHC Clas Is Presentation 637. 637. 637. id="p-637" id="p-637" id="p-637" id="p-637" id="p-637" id="p-637" id="p-637" id="p-637" id="p-637" id="p-637"
[637] Certa embodin imen ofts the cell-target moleculeing ofs the prese ntinvention are cytotoxic becaus theye comprise a CD8+ T-cell epitope capable of being delivered to the MHC clas Is presentati pathwaon 7 ofy a target cell and presented on a cellular surface of the targe cellt For. example, T-cell epitope delivering, CD8+ T- cell hyper-immunized, Shiga toxin effector polypeptides of the prese ntinvention, wit hor without endogenous epitope de-immunization, may7 be used as components of cell-target molecing ules for applications involv ingindirec cellt -killing. 638. 638. 638. id="p-638" id="p-638" id="p-638" id="p-638" id="p-638" id="p-638" id="p-638" id="p-638" id="p-638" id="p-638"
[638] In certa embodimein nts of the cell-targeti moleculeng ofs the present invention, upon contacti a ngcell phy sically coupled with an extracel targelular t biomolecul of ethe cell-target bindinging region the, cell-target moleingcule of the invention is capabl ofe indirectl causy ing the death of the cell such, as, e.g., via the presentation of one or more T-cell epitop esby the targ cellet and the subsequent recruitme ofnt CTLs whic hkill the targe cell.t 639. 639. 639. id="p-639" id="p-639" id="p-639" id="p-639" id="p-639" id="p-639" id="p-639" id="p-639" id="p-639" id="p-639"
[639] The presentat ofion an antigenic peptide complexed with a MHC clas I s molecule by a cell sensitizes the presenting cell to targe tedkilling by cytotoxic T- cel ls(CTLs) via the induction of apoptosis lysis,, and/or necrosis In. addition, theWO 2016/196344 PCT/US2016/034778 233 CTLs which recognize the targe cellt may release immuno-stimulator cytokines,y such as, e.g., interfer gammaon (IFN-gamma tumor), necrosi facs tor alpha (TNF), macrophage inflammatory protein-1 beta (MIP-lbeta and), interleukins such as IL- 17, IL-4, and IL-22. Furthermor CTLse, activa tedby recognition of a presente d epitope may indiscriminate kill lyothe cellsr proximal to the presenting ceil regardless of the peptide-MHC class I complex repertoir presee nted by those proximal cells (Wiedemann A et al., Proc Natl Acad Set USA 103: 10985-90 (2006)). 640. 640. 640. id="p-640" id="p-640" id="p-640" id="p-640" id="p-640" id="p-640" id="p-640" id="p-640" id="p-640" id="p-640"
[640] Because of MHC allele diversi withinty differ entspecies, a cell-targeting molecule of the prese ntinvention comprisi ngonly a single epitope may exhibit varie effd ectiveness to differ entpatien orts subjects of the same species However,. certain embodiments of the cell-target moleculeing ofs tire present invention may eac hcompris multie ple, T-cell epitopes that are capable of being deliver toed the MHC class I system of a target cell simultaneously. Thus, for certa embodimin ents of the cell-targeti moleculeng ofs the prese ntinvention, a. cell-targeti molengcule is used to trea diffet rent subjects with considerable difference in theirs MHC molecule’ epits ope-pepti bindinde affg initi (i.ees. considerable difference in theirs MHC alleles and/o MHCr genotype Ins). addition, certain embodiments of the cell- targeting molecules of the prese ntinvention reduce or prevent targ ceetil adaptations to escape killing (e.g. a target cance cerll mutating to escape therapeutic effectiveness or "mutant escape") by using multiple cell-killi mechang nisms simultaneously (e.g. direc killt ing and indirec killingt via multiple different T-cell epitopes simultaneously). 2, Direct Cell-Kill via Cell-Targeted Shiga, Toxin Cytotoxicity 641. 641. 641. id="p-641" id="p-641" id="p-641" id="p-641" id="p-641" id="p-641" id="p-641" id="p-641" id="p-641" id="p-641"
[641] Certa embodin imen ofts the cell-target moleculeing ofs the prese ntinvention are cytotoxic becaus theye comprise a catalytica actllyive, Shiga toxin effector polypeptide and regardles of thes presence of an immunogenic, CD 8+ T-cell epitop e in the molecule For. example, CD8+ T-cell hyper-immunize Shigad, toxin effector polypepti desof the present invention, with or without endogenous epitope de- immunization, may be used as components of cell-targetin moleculesg for applications involving direc cell-killt suching, as, e.g., via the ribotoxic, enzymat ic activity of a Shiga toxin effector polypeptide or ribosome bindin gand interference with ribosome function due to a non-cata lyticmechanism(s).[642] For certain embodiment ofs the CD8+ T-cell hyper-immunize celld,­ target moleing cule ofs the prese ntinvention, upon contact aing cel physil call y coupled wit han extracell targetular biomolecule of the cell-targeti bindingng region , the cell-targeti molengcule of the invention is capabl ofe direct causily ng the deat h of the cel l,such as, e.g., without the involvem ofent an untarget cytoed, toxi T-celc l (see Section V-D, supra).
G. Select iveCytotoxicit amongy Cell Types 643. 643. 643. id="p-643" id="p-643" id="p-643" id="p-643" id="p-643" id="p-643" id="p-643" id="p-643" id="p-643" id="p-643"
[643] Certa cellin -target moleingcule ofs the present invention have uses in the selecti killve ing of specifi tarc get cell ins the presence of untarget bystaed, nder cells.
By targeti theng delivery of Shiga toxin effect polypeptidesor of the present inventio to nspecifi cellc vias a cell-targeti bindingng region(s) the, cell-targeti ng molecules of the present inventio cann exhibi cellt -type specific, restrict celedl-ki ll activiti resultes ingin the exclusi veor preferent killialing selected cell types in the presence of untargeted cell s.Similarly, by target theing delivery of immunogenic T- cel epitl opes to the MHC clas I spathway of target cell thes, subsequent presenta tion of T-cell epitopes and CTL-mediat edcytolysi ofs target cells induced by the cell­ targeti moleng cule ofs the invention can be restricted to exclusively or preferent ially killing select celed typesl in the presence of untarge cellted s.In addition, both the cell-target deliedvery of a cytotoxic, Shig atoxin effector polypepti regionde and an immunogenic, T-cell epitope can be accomplished by a single cell-target ing molecule of the present invention such that deliver of both potentially cytotoxic components is restricted exclusively or preferenti toall targety cell ins the presenc e of untarget celled s. . 644. 644. 644. id="p-644" id="p-644" id="p-644" id="p-644" id="p-644" id="p-644" id="p-644" id="p-644" id="p-644" id="p-644"
[644] For certai embodimn ents, the cell-targeti molengcule of the present invention is cytotoxic at certain concentrati Inons. certain embodiments, upon administration of the cell-targeting molecule of the present invention to a mixture of cell types, the cytotoxi cell-tc argeti molngecul is ecapabl ofe selectively killing those cell whichs are physicall coupledy wit han extracell targetular biomolec ulecompared to cell types not physical coupledly wit han extracell targetular biomolecul Fore. certai n embodiments the ,cytotoxic cell-targeting molecule of the present inventio is n capable of selectively or preferenti causiallyng the deat ofh a specific cel typel within a mixture of two or more different cell types This. enable targets ing cytotoxic activity to specific cell types wit ha high preferenti ality,such as a 3-fold -234-WO 2016/196344 PCT/US2016/034778 235 cytotoxic effect, over "bystander" ceil types that do not expre ssthe target biomolecule. Alternatively the expr, ess ionof the targe biomolecult of ethe binding region may be non-exclusi tove one cell type if the targe biomolecult is expree ss ed in low enoug amountsh and/o physicr all coupledy in low amounts with cell Apes that are not to be target ed.This enables the targeted cell-killing of specif icceil types with a high preferentia suchlity, as a 3-fold cytotoxic effect, over "bystander" cell types that do not express significant amount ofs the targ biomoleculet or eare not physica llycoupled to significant amounts of the targe biomolet cule. 645. 645. 645. id="p-645" id="p-645" id="p-645" id="p-645" id="p-645" id="p-645" id="p-645" id="p-645" id="p-645" id="p-645"
[645] For certain further embodiment upons, administration of the cytotoxic ceil- targeting molecule to two differ entpopulations of cell type s,the cytotoxic cell- targeting molecule is capabl ofe causing ceil deat ash defined by the half-maxima l cytotoxic concentrat (CD50)ion on a population of target cell whoses, members express an extracel targelular biomolecult of ethe bindin regiog ofn the cytotoxic cell-target moleculeing at a, dose at least three-time lowers than the CD50 dose of the same cytotoxic cell-targeting molecule to a population of cells whose members do not expre ssan extracel targelular biomoleculet of the bindin regg ion of the cytotoxic cell-target molecing ule. 646. 646. 646. id="p-646" id="p-646" id="p-646" id="p-646" id="p-646" id="p-646" id="p-646" id="p-646" id="p-646" id="p-646"
[646] For certain embodiments, the cytotoxic activity of a cell-targeti moleculeng of the prese ntinvention towar populad tions of cell types physicall coupledy with an extracellular targe biomolecult is ate least 3-fold higher than the cytotoxic activity towar populatid ons of cell types not physically coupled wit hany extracellular target biomolecul of ethe binding region. According to the prese ntinvention, selecti ve cytotoxici mayty be quantifi ined terms of the ratio (a/b) of (a) cytotoxicity toward s a population of cells of a specifi cellc type physically coupled with a target biomolecul of ethe binding regio ton (b) cytotoxic towardsity a population of cel lsof a cel typel not physically coupled with a targ biomoleculet ofe the binding region.
In certai embodimen nts, the cytotoxi ratiocity is indicative of selective cytotoxi city which is at leas 3-fot ld, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold 30-fold,, 40-fold, 50-fold, 75-fold, 100-fold, 250-fold, 500-fold, 750-fold, or 1000-fol higherd for populations of ceils or cell types physicall coupledy with a targ biomolecet uleof the binding region compared to populations of cells or cell types not physically coupled with a targ biomoleculet of ethe binding region. 647. 647. 647. id="p-647" id="p-647" id="p-647" id="p-647" id="p-647" id="p-647" id="p-647" id="p-647" id="p-647" id="p-647"
[647] For certain embodiment thes, preferential cell-killing function or selecti ve cytotoxici of tya cell-targeti molengcule of the prese ntinvention is due to an WO 2016/196344 PCT/US2016/034778 236 additional exogenou mates rial (e.g. a cytotoxic material and/or) heterologous T-cell, epitope present in a Shiga toxin effecto polypeptr ideof the prese ntinvention and not necessa rilya result of the catalyti actcivit ofy a Shiga toxin effecto polypeptider region. 648. 648. 648. id="p-648" id="p-648" id="p-648" id="p-648" id="p-648" id="p-648" id="p-648" id="p-648" id="p-648" id="p-648"
[648] This preferential cell-kil lingfunction allow a targes tedcell to be kille byd certain cytotoxic, cell-target moleculesing of the prese ntinvention under varie d conditions and in the presence of non-targeted bystander cells, such as ex vivo manipula tedmixture ofs ceil types in, vitro cultured tissues wit hmixture ofs cel l type s,or in vivo in the presence of multiple cell types (e.g. in situ or in a native location withi an multicellul organism).ar H. Deliver ofy Additional Exogenous Materi intoal the Interior of Targeted Ceil s 649. 649. 649. id="p-649" id="p-649" id="p-649" id="p-649" id="p-649" id="p-649" id="p-649" id="p-649" id="p-649" id="p-649"
[649] In addition to cytotoxic, cytosta andtic immune, stimulat applicion ations , cell-target moleculeing ofs the present invention optional mayly be used for targete d intracell deliularvery7 functions, such as, e.g., in applications involving information gathering and diagnost funcic tions. 650. 650. 650. id="p-650" id="p-650" id="p-650" id="p-650" id="p-650" id="p-650" id="p-650" id="p-650" id="p-650" id="p-650"
[650] Because the cell-targetin m oleculg es of the invention, including reduced cytotoxici and/orty nontoxi formc thers eof, are capable of entering cells physicall y coupled with an extracellular targe biomolect ulerecogniz byed the cell-targeti ng molecule’s binding region, certai embodimn ents of the cell-targetin moleculesg of the invention may be used to deliver additional exogenou mats eri alsinto the interior of targete celld type s.For example, non-toxic variant of thes cytotoxic, cell- targeting molecules of the invention, or optionall cytotoxicy variants may, be used to deliv eradditional exogenou maters ials to and/or labe thel interior of scel ls physicall coupley withd an extracel targelular biomt olec uleof the bindin gregio ofn the cell-targeti molecng ule. Various type ofs ceils and/o cer ll populations which express target biomolecules to at least one cellular surface may be targe tedby the cell-target moleculeing ofs the invention for receiving exogenou materials Thes. functional compone ntsof the prese ntinvention are modular in ,that various Shiga, toxin effector polypeptide additis, onal exogenou mates rial ands, bindin gregions may be associated wit heac hothe tor provide cell-targeti molecng ules suitable for divers apple ication involvs ing cargo delivery7, such as, e.g., non-invasi inve, vivo imaging of tumor cells.WO 2016/196344 PCT/US2016/034778 237 651. 651. 651. id="p-651" id="p-651" id="p-651" id="p-651" id="p-651" id="p-651" id="p-651" id="p-651" id="p-651" id="p-651"
[651] This delivery of exogenou mates rial function of certa cellin -targetin g molecules of the prese ntinvention may be accomplished under varied conditions and in the presence of non-target bystandered cells such,, as, e.g., an ex vivo manipulated targe celt l,a targe cetll cultured in vitro, a targe cellt within a tissue sample cultured in vitro, or a targe ceilt in an in vivo setting like withi an multicellul organiar sm. Furthermo there, selecti deliveryve of exogenou materials to certain cel lsby certa cell-in target moleculeing ofs the present invention may be accomplished under varie condid tions and in the presence of non-targeted bystande r cell suchs, as ex vivo manipula tedmixtur esof cell types in, vitro cultured tissues with mixture ofs cell type s,or in vivo in the presence of multiple cell types (e.g. in situ or in a native location within a multicellul organism).ar 652. 652. 652. id="p-652" id="p-652" id="p-652" id="p-652" id="p-652" id="p-652" id="p-652" id="p-652" id="p-652" id="p-652"
[652] Shiga toxin effecto polyper ptide ands cell-target molecing ules whic hare not capable, such as a certai concentn rat rangesion of, killing a targ ceetll and/or delivering an embedded or inserted epitope for cell-surfa presentace tionby a MHC molecule of a target cell may still be usefill for delivering exogenous materials into cell suchs, as, e.g., detect ionpromoti agents.ng 653. 653. 653. id="p-653" id="p-653" id="p-653" id="p-653" id="p-653" id="p-653" id="p-653" id="p-653" id="p-653" id="p-653"
[653] For certa embodimein nts, the Shiga toxin effector polypepti desof the present invention exhibits low to zero cytotoxicity' and thus are referre to dhere inas "noncytotoxic and/or reduced cytotoxic." For certain embodiments, the cell- targeting molecule of the present invention exhibits low to zero cytotoxici andty may be referred to as "noncytot"oxic and/o "rreduc edcytotoxic variants" For. example, certain embodiments of the molecules of the prese ntinvention do not exhibit a significant leve ofl Shiga toxin based cytotoxi wherecity atin doses of less than 1000 nM, 500nM, 100 nM, 75 nM, 50 nM, there is no significant amount of cell death as compar edto the appropriate refere ncemolecule, such as, e.g., as measured by an assa knowny to the skilled worker and/o descr ribed herei n.For certain further embodiments, the molecule ofs the prese ntinvention do not exhibit any toxicity■' at dosage ofs 1-100 pg per kg of a mammalia recn ipient. Reduced- cytotoxic variants may still be cytotoxic at certai concen ntra ortions dosages but exhibit reduced cytotoxici suchty, as, e.g., are not capable of exhibiti nga significa nt leve ofl Shiga toxin cytotoxi incit cery tai situatn ions. 654. 654. 654. id="p-654" id="p-654" id="p-654" id="p-654" id="p-654" id="p-654" id="p-654" id="p-654" id="p-654" id="p-654"
[654] Shiga toxin, effecto polyper ptide of thes prese ntinvention, and certa cell-in targeting molecules comprising the same, can be rendered non-cytotoxi suchc, as, e.g., via the addition of one or more amino aci dsubstitutions known to the skilled WO 2016/196344 PCT/US2016/034778 238 worker to inactive a Shiga toxin A Subunit and/o Shigar toxin effector polypeptide, including exemplary substitutions described herei n.The non-cytoto andxic reduced cytotoxic variant of thes cell-target moleculeing ofs the prese ntinvention may be in certain situations more suitable for delivery of additional exogenou mats eri alsthan more cytotoxic variants.
Informati Gatheon ring for Diagnosti Functionsc 655. 655. 655. id="p-655" id="p-655" id="p-655" id="p-655" id="p-655" id="p-655" id="p-655" id="p-655" id="p-655" id="p-655"
[655] In certa ceinll-targeti moleculeng ofs the present invention have uses in the in vitro and/o inr vivo detect ionof specif iccells, cell types and/o, cer ll populatio ns, as wel asl specif icsubcellula compar rtme ofnts any of the aforementione d.
Reduced-cytotoxicity and/or nontoxi formc ofs the cytotoxic, cell-targetin g molecules of the invention that are conjugated to detectio promotn ing agents optionally may be used for diagnostic functions, such as for companion diagnostic s used in conjunction wit ha therapeutic regimen comprising the same or a relate d binding region, such as, e.g., a binding regio withn high-affmity binding to the same targe biomolect ule, an overlapp epiting ope, and/or the same epitope. 656. 656. 656. id="p-656" id="p-656" id="p-656" id="p-656" id="p-656" id="p-656" id="p-656" id="p-656" id="p-656" id="p-656"
[656] In certain embodiments, the cell-target moleculeing descs ribe hereind are used for both diagnosi ands treatmen or fort, diagnos alone.is When the same cytotoxic cell-targeting molecule is used for both diagnos andis treatment, for certain embodiments of the present invention tire cell-target moleingcule variant which incorpor atea detects ionpromoti agentng for diagnos mayis have its cytotoxic reducedity or may be rendere nontoxicd by catalyti inactc ivati ofon its Shiga toxin effector polypeptide region(s via) one or more amino acid substitutions, including exemplary substitutions described herei n.For example, certa nontoxiin c variant of thes cell-target moleculesing of the prese ntinvention exhibit les thans 5%, 4%, 3%, 2%, or 1% death of targe cellst after administrat ofion a dose les sthan 1 mg/kg. Reduced-cytotoxicity variant mays still be cytotoxic at certain concentra ortions dosages but exhibi reduct edcytotoxi city,such as, e.g., are not capable of exhibiting a significant leve ofl Shiga toxin cytotoxicity as described herein. 657. 657. 657. id="p-657" id="p-657" id="p-657" id="p-657" id="p-657" id="p-657" id="p-657" id="p-657" id="p-657" id="p-657"
[657] Ilie ability to conjugate detectio promon ting agents known m the art to various cell-targeti molecng ules of the present invention provides useful compositions for the detect ionof certain cells such, as, e.g., cancer, tumor immun, e, and/o infer cte cellsd These. diagnos embodimtic ents of the cell-targetin molecg ules WO 2016/196344 PCT/US2016/034778 239 of the invention may be used for informati gatherion viang various imaging techniques and assays known in the art. For example, diagnosti embodc imen ofts the cell-targeti molecng ules of the invention may be used for informati gatheon ring via imaging of intracell organellular (e.ges .endocytoti Golgi,c, endoplasmic reticulum, and cytosolic compartme ofnts) individual cance celr ls, immune cell s, and/o infer cte cellsd in a patient or biopsy sample. 658. 658. 658. id="p-658" id="p-658" id="p-658" id="p-658" id="p-658" id="p-658" id="p-658" id="p-658" id="p-658" id="p-658"
[658] Various types of informati mayon be gathered using the diagnostic embodiments of the cell-target molecing ules of the invention whether for diagnos tic uses or othe usesr .This informati mayon be useful, for example, in diagnosing neoplas celltic types determining, therapeutic susceptibiliti ofes a patient’s disease, assaying the progression of anti-neoplast theraic pies overtime ass, aying the progress ofion immunomodulator therapiey overs time ass, aying the progression of antimicrobial therapies over time eval, uating the presenc ofe infecte cellsd in transplantation materia evaluatils, theng presence of unwanted cell types in transplantation materia and/ols, evaluatingr the presence of residual tumor cells after surgical excision of a tumor mass. 659. 659. 659. id="p-659" id="p-659" id="p-659" id="p-659" id="p-659" id="p-659" id="p-659" id="p-659" id="p-659" id="p-659"
[659] For example, subpopulations of patients might be ascerta inedusing information gathered using the diagnosti variantc of sthe cell-targeting molecules of the invention, and then individual patients coul bed furthe catr egor izeintod subpopulations based on thei uniquer characteris ticreveal(®) usinged those diagnosti embodiments.c For example, the effectiveness of specifi pharmaceutic cals or therapi mightes be a criter ionused to define a. patient subpopulati on.For example, a nontoxic diagnos vartic iant of a particular cytotoxic, cell-targetin g molecule of the invention may be used to differentiat whiche patien arets in a cla ss or subpopulation of patients predict toed respond positive toly a cytotoxic varia ofnt that cell-targetin molegcule of the invention. According assocly, iated methods for patient identificat patiion,ent stratifica andtion, diagnosis using cell-targetin g molecules of the prese ntinvention, including non-toxic variants of cytotoxic, cell- targeting molecules of the prese ntinvention, are considere to dbe withi then scope of tiie prese ntinvention. 660. 660. 660. id="p-660" id="p-660" id="p-660" id="p-660" id="p-660" id="p-660" id="p-660" id="p-660" id="p-660" id="p-660"
[660] Tire express ionof the target biomolec uleby a cell need not be native in order for cell-targetin by a cellg -targetin molegcule of the present invention, such as, e.g., for direc cell-kill,t indirec cellt -kil deliveryl, of exogenous materi alslike T-cell epitopes and/or, informati gatherion ng.Cell surfa ceexpress ionof the targe tWO 2016/196344 PCT/US2016/034778 240 biomolecul coulde be the resul oft an infection, the presence of a pathogen and/, or tiie presence of an intracellula microbiar pathogen.l Expression of a target biomolecul coulde be artific suchial as, for exampl bye, force ord induced express ionafter infection wit ha vira expressl ionvector, see e.g. adenoviral, adeno- associated viral and, retroviral systems. An example of inducing express ionof a targe biomolecult is thee upregulation of CD38 express ionof cells expose tod retinoids, like a\\-trans retinoi acidc and various synthe retinoids,tic or any retinoi c acid receptor (RAR) agonis (Drat ch J et ah, Cancer Res 54: 1746-52 (1994); Uruno A et al., J Leukoc Biol 90: 235-47 (2011)). Expression of CD30 can be induced in both B-cells and T-cel lsby exposur toe by mitogens, phytohemaggluti (PHninA), staphylococcal protei A,n EBV virus human, T-cell leukemi virusa 1 or 2 (HTLV-1 0rHTLV-2) (see e.g. Stei nH et al., Blood 66; 848-58 (1985)). In another example, CD20, HER2, and EGFR express ionmay be induced by exposing a cell to ionizing radiati (Waon ttenbe Mrg et al., Br J Cancer 110: 1472-80 (2014)). Further, PSMA expression is upregulated in response to androgen deprivation (see e.g. Chang S et al., Cancer 88: 407-15 (2000); Metier B et al., EJNMMI Res 5: 66 (2015)).
VI. Production, Manufact ure,and Purification of Shiga Toxin Effect Polypeptor ides of the Invention and Cell-Targeting Molecul Comprisinges the Same 661. 661. 661. id="p-661" id="p-661" id="p-661" id="p-661" id="p-661" id="p-661" id="p-661" id="p-661" id="p-661" id="p-661"
[661] The Shiga toxin effector polypepti desand certain cell-target moleculeing ofs the prese ntinvention may be produce usingd techniques wel knownl to those of skill in the art. For example, Shiga toxin effector polypepti desand ceil-targeting molecules of the invention may be manufactur byed standa syntheticrd methods, by use of recombina exprnt ess ionsystems or, by any oilier suitable metho d.Thus, Shiga, toxin effector polypeptides and cell-targeti molecng uies of the invention may be synthesized in a number of ways including,, e.g. methods comprising: (1) synthesizing a polypept ideor polypeptide component of a cell-targeti moleculeng using standar solid-phased or liquid-phas methodology,e either stepwise or by fragment assembly, and isolating and purifying the fina polypeptidel compound produc (2)t; expressing a polynucleotid that encodese a protein or prote compoin nent of a cell-targetin molegcule of the invention in a host cell and recover theing express ionproduc fromt the host cell or host cell cultur ore; (3) cell-free, in vitro express ionof a polynucleotid encodinge a polypeptide or polypeptide component of a cell-targetmg molecule of the invention, and recovering the express ionproduc ort; WO 2016/196344 PCT/US2016/034778 241 by any combinati ofon the methods of (1), (2) or (3) to obtain fragmen ofts the protei component,n subsequently joining (e.g ligating) the peptide or polypept ide fragmen tots obtain a polypeptide compone nt,and recover theing polypept ide component. 662. 662. 662. id="p-662" id="p-662" id="p-662" id="p-662" id="p-662" id="p-662" id="p-662" id="p-662" id="p-662" id="p-662"
[662] It may be preferable to synthes aize Shiga toxin effector polypeptide of the present invention, cell-target moleculeing of the prese ntinvention, or a protei n component of a cell-targeti molengcule of the invention by means of solid-phase or liquid-phas peptidee synthes is.Polypeptides and cell-targeti moleculeng ofs the present invention may suitably be manufactured by standar syntheticd methods .
Thus, peptides may be synthesize by,d e.g. methods comprisin synthesg izing the peptide by standa solidrd -phas or eliquid-phase methodology, either stepwise or by fragment assembly, and isolating and purifying the final peptide product. In this context ref, ere ncemay be made to WO 1998/011125 or, inter alia, Fields G et al., Principles and Practice of Solid-Phase Peptide Synthesis (Synthe ticPeptides Grant, G, ed., Oxford Universit Press,y U.K., 2nd ed., 2002) and the synthesis examples therein. 663. 663. 663. id="p-663" id="p-663" id="p-663" id="p-663" id="p-663" id="p-663" id="p-663" id="p-663" id="p-663" id="p-663"
[663] Shiga toxin effecto polypeptidesr and cell-target molecing ules of the present invention may be prepar ed(produced and purified) using recombina technint ques wel knownl in the art. In general, methods for preparing protei byns culturing host cel lstransforme or transd fecte withd a vector comprisin theg encoding polynucleotid and purife ying or recover theing protei fromn cell culture are describe in,d e.g., Sambrook J et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laborat oryPress NY,, U.S., 1989); DiefTenbac Ch et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press N.Y.,, U.S., 1995). Any suitable host cel mayl be used to produc ae polypeptide and/o cellr - targeting protei ofn the invention. Host ceils may be ceils stably or transiently transfecte transford, med,transduced or infected with one or more express ionvectors w'hich drive express ionof a polypeptide of the invention. In addition, a Shiga toxin effector polypeptide and/or cell-target moleingcule of the invention may be produced by modifying the polynucleotid encodinge a polypeptide or cell-targetin g protei ofn the invention that resul int alter ingone or more amino acids or deleting or inserting one or more amino acid ins order to achie vedesired properti suches, as change cytod toxic changeity, cytosd tati effecc ts,and/or change serd um half-life.WO 2016/196344 PCT/US2016/034778 242 664. 664. 664. id="p-664" id="p-664" id="p-664" id="p-664" id="p-664" id="p-664" id="p-664" id="p-664" id="p-664" id="p-664"
[664] There are a wide variety of express ionsystems which may be chosen to produc ae polypeptide or cell-targeti proteing ofn the present invention. For example, host organisms for express ionof cell-targeti proteinsng of the invention include prokaryote suchs, as E. coU and B. subtilis, eukaryotic cells such, as yeas t and filamento fungius (like S. cerevisiae. P. pastoris, A. awamori, and K. lactis\ algae (like C. reinhardtiiY insect cell lines, mammalia cellsn (like CHO cells), plant cell lines and, eukaryotic organisms such as transgenic plants (like A. thaliana and N. bentham land). 665. 665. 665. id="p-665" id="p-665" id="p-665" id="p-665" id="p-665" id="p-665" id="p-665" id="p-665" id="p-665" id="p-665"
[665] Accordingly the prese, ntinvention als provideso methods for producing a Shiga toxin effector polypeptid e and/or cell-targetin molegcule of the present invention accordi tong above recite metd hods and using a polynucleotid enceoding part or all of a poh'peptide of die invention or a protein componen oft a cell- targeting protei ofn the invention, an express ionvector comprisin atg leas onet polynucleotid of thee invention capable of encoding part or all of a polypeptide or cell-targetin proteig ofn the inventi onwhen introduced into a. host cel l,and/or a. host cell comprisin a gpolynucleot oride express ionvector of the invention. 666. 666. 666. id="p-666" id="p-666" id="p-666" id="p-666" id="p-666" id="p-666" id="p-666" id="p-666" id="p-666" id="p-666"
[666] When a protein is expressed using recombina technt nique in sa host cell or cell-f reesystem it ,is advantage toous separate (or purify) the desired protei awan y from odier components such, as host cell factors in order, to obtain preparation that s are of high purity or are substantial homogeneouly Purifs. ication can be accomplis hedby methods well known in the art, such as centrifugation techniques , extrac tiontechniqu chromes, atogr andaphic fractionation techniques (e.g .size separation by gel filtration, charge separat byion ion-exchange colum n,hydrophobi c interact chroion matogr reveaphy,rse phase chromatography, chromatography on silica, or cation-exchan resinsge such as DEAE and the like, chromatofoc usinand g, Protein A Sepharose chromatogra to phy'remove contamina nts)and precipi, tati on techniques (e.g. ethano precl ipitation or ammonium sulfate precipitation). Any number of biochemic purificatioal techniquesn may be used to increas thee purif yof a polypeptide and/o cer ll-targeti moleculng ofe the prese ntinvention. In certai n embodiments, the polypeptides and cell-target molecing ules of the invention may' optionall be purify ied in homo-multim ericforms (e.g. a molecular complex comprising two or more polypeptides or cell-targeti molecng ules of the invention). 667. 667. 667. id="p-667" id="p-667" id="p-667" id="p-667" id="p-667" id="p-667" id="p-667" id="p-667" id="p-667" id="p-667"
[667] In the Examples below are descriptions of non-limit ingexamples of methods for produci ngexemplary, Shiga toxin effecto polypeptidesr and cell-targetin gWO 2016/196344 PCT/US2016/034778 243 molecules of die present invention, as well as specif icbut non-limiti aspectng ofs production methods.
VIL Pharmaceutica and Diagnostil Compositionsc Comprising Cell-Targeting Molecul ofes the Present Invention 668. 668. 668. id="p-668" id="p-668" id="p-668" id="p-668" id="p-668" id="p-668" id="p-668" id="p-668" id="p-668" id="p-668"
[668] The present invention provides Shiga toxin effector polypeptides and cell- targeting molecules for use ,alone or in combinat ionwit hone or more additional therapeutic agents, in a pharmaceutic composition,al for treatme ornt prophylaxis of conditions, disease disordes, orrs, symptoms describe ind further detail below (e.g. cancers, malignant tumors non-m, aligna tumorsnt growth, abnormalities immu, ne disorde andrs, microbial infections) Tire. present invention further provide s pharmaceuti compocal sitions comprisin a gShiga toxin polypeptide or cell-targeting molecule of tire present invention, or a pharmaceutica acceptally sablelt or solvate thereof, accordi tong the invention, together with at leas onet pharmaceuticall y accepta cablerrier, excipien ort, vehicle In. certa embodin iment thes, pharmaceuti cal composition of the present invention may comprise homo-multim ericand/o heterr o- multimer formsic of a Shiga toxin effector polypeptides or cell-targetin molegcule of the prese ntinvention. The pharmaceut compical ositions of the invention are usefi ll in methods of treati ng,ameliorat oring, preventing a disease, condition, disorder, or symptom described in furthe detailr below. Each such disease, condition, disorder , or symptom is envisioned to be a separa embodte iment with respect to uses of a pharmaceutica compositionl accordi tong the invention. The invention further provide pharmas ceut compoical sitions for use in at leas onet method of treatment according to the invention, as describe ind more detail below. 669. 669. 669. id="p-669" id="p-669" id="p-669" id="p-669" id="p-669" id="p-669" id="p-669" id="p-669" id="p-669" id="p-669"
[669] As used herein, the term "spatient" and "subject"' are used interchangeably to refe tor any organism, commonly vertebra suchtes as humans and animals, which present sympts oms, signs, and/o indicr atio ofns at leas onet disease, disorder, or condition. These term includs mame mal suchs as the non-limit ingexamples of primates lives, tock animals (e.g. cattle hors, es pigs, ,sheep, goat etc.),s, compani on animals (e.g. cat s,dogs, etc?) and laboratory animals (e.g. mice ,rabbits, rats, etc.). 670. 670. 670. id="p-670" id="p-670" id="p-670" id="p-670" id="p-670" id="p-670" id="p-670" id="p-670" id="p-670" id="p-670"
[670] As used herein, "treat" "trea, ting," or "treatment" and grammat icalvariants thereof refer to an approa chfor obtaining beneficia or ldesired clinical result Thes. term mays refe tor slowing the onset or rate of developme ofnt a condition, disorder or disease, reducing or alleviating symptoms associa tedwith it, generating a WO 2016/196344 PCT/US2016/034778 244 complete or parti regral ession of the condition, or some combinat ionof any of the above. For the purposes of this invention, benefici oral desired clinica resultl s include but, are not limite to,d reduction or alleviation of symptoms, diminishmen t of extent of disease, stabilization (e.g. not worsening) of sta teof disease, delay or slowing of diseas progre ess ion,amelioratio or palliatn ofion the diseas stae te, and remission (whethe partir oral total whether), detectable or undetectable "Treat. " , "treating," or "treatment" can also mean prolonging surviva relatl iveto expect ed surviva timel if not receiving treatment. A subjec (e.g.t a human) in need of treatment may thus be a subject already afflict withed the diseas ore disorde m r question. 'The term "streat" "trea, ting," or "treatment" includes inhibition or reduction of an increas ine severity of a pathologic staal teor symptoms relati tove the absence of treatm ent,and is not necessar meaily nt to imply complete cessation of the relevant disease, disorder, or condition. With regard to tumors and/or cance rs, treatme includent reductis inon overall tumor burden and/or individual tumor size. 671. 671. 671. id="p-671" id="p-671" id="p-671" id="p-671" id="p-671" id="p-671" id="p-671" id="p-671" id="p-671" id="p-671"
[671] As used herein, the terms "prevent,"' "preventing," "prevention" and grammatica variantl thereofs refe tor an approach for preventing the developme of,nt or altering the pathology of, a condition, disease, or disorder. According ly, "preventi"' onmay refer to prophylact or preveic ntive measures. For the purposes of this invention, benefici oral desired clinical results include, but are not limited to, prevention or slowi ngof symptoms, progress orion developme ofnt a disease, whether detectable or undetectabl A. e.subject (e.g. a human) in need of prevention may thus be a subject not yet afflicted with the disease or disorder in question. Tire ter m"preventi" onincludes slowing the onset of diseas rele ati tove the absenc ofe treatment, and is not necessa rilymeant to imply permanent prevention of the relevant diseas disore, der or condition. Tirus "preventi" ngor "prevention" of a condition may in certa contextsin refe tor reducin theg risk of developi theng condition, or preventing or delaying the developmen of symt ptoms associa tedwith the condition. 672. 672. 672. id="p-672" id="p-672" id="p-672" id="p-672" id="p-672" id="p-672" id="p-672" id="p-672" id="p-672" id="p-672"
[672] As used herein, an "effecti amountve " or "therapeuticall effectivey amount"' is an amount or dose of a composition (e.g. a therapeutic composition, compound, or agen thatt) produce ats leas onet desired therapeutic effec int a subject, such as preventing or treating a target condition or beneficially alleviating a sympto m associated with tire condition. The most desirabl therapee utically effective amount is an amount that will produce a desired efficacy of a particula treratmen selectet dWO 2016/196344 PCT/US2016/034778 245 by one of skill in the art for a give nsubject in need thereof This. amount will vary depending upon a variet ofy factors understood by the skilled worke includingr, but not limited to the characteristics of the therapeutic composition (including activity, pharmacokinet pharmacics, odynamic and bioavs, ailability), the physiologica l condition of the subject (including age sex,, diseas type,e diseas stae ge, genera l physical condition, responsive nessto a give ndosage, and type of medication), the natur ofe the pharmaceutically accepta carrible orer carriers in the formulation, and tire route of administration. One skilled in the clinic andal pharmacologica arts willl be able to determi ane therapeutica effectivelly amount throug routineh experimentat namelyion, by monitoring a subject’s response to administrati of aon composition and adjusting the dosage according (seely e.g. Remington: The Science and Practice ofPharmacy (Genna roA, ed., Mack Publishing Co., Easton, PA, U.S., 19th ed., 1995)). 673. 673. 673. id="p-673" id="p-673" id="p-673" id="p-673" id="p-673" id="p-673" id="p-673" id="p-673" id="p-673" id="p-673"
[673] Di agnos ticcompos onsiti of the prese ntinvention compris ae cell-targeting molecule of the prese ntinvention and one or more detection promoting agents.
When producing or manufactur aing diagnost composiic tion of the present invention, a cell-targeti moleculeng of the prese ntinvention may be direct orly indirectl linkedy to one or more detectio promotin agents.ng There are numerous standar technid ques known to tire skilled worke forr incorpora ting,affixing, and/o r conjugating various detection promoti agentsng to proteins or proteinaceo us compone ntsof molecules, especially to immunoglobulins and immunoglobul in- derived domains. 674. 674. 674. id="p-674" id="p-674" id="p-674" id="p-674" id="p-674" id="p-674" id="p-674" id="p-674" id="p-674" id="p-674"
[674] There are numerous detectio promotn ing agents known to the skill edworker, such as isotopes, dyes, colorimetr ageicnts, contrast enhanci ageng nts, fluoresc ent agents, bioluminescent agents, and magneti agenc ts, which can be operably linked to the polypeptides or cell-targeti molecng ules of the invention for informati on gather mething ods, such as for diagnost and/oric prognostic applications to diseases , disorde orrs, conditions of an organism (see e.g. Cai W et al., JNuclMed 48: 304-10 (2007); Nayak T, Brechbiel M, Bioconjug Chern 20: 825-41 (2009); Paudya Pl et al., Oncol Rep 22: 115-9 (2009); Qiao J et al., PLoS ONE 6: 618103 (2011); Sano K et al., Breast Cancer Res 14: R61 (2012)). Thes eagents may be associated with, linke to,d and/o incorporar withinted the polypeptide or cell-targetin molecg ule of the invention at any suitable position. For exampl thee, linkage or incorporation of the detect ionpromoti agenng mayt be via an ammo acid residue( ofs) a molecule of WO 2016/196344 PCT/US2016/034778 246 tiie prese ntinvention or via some type of linkage known in the art, including via linker and/ors chelators The. incorporati of theon agent is in such a way to enable the detect ionof the presence of the diagnost composiic tion in a screen, assay, diagnos procetic dure, and/or imaging technique. 675. 675. 675. id="p-675" id="p-675" id="p-675" id="p-675" id="p-675" id="p-675" id="p-675" id="p-675" id="p-675" id="p-675"
[675] Similarly, there are numerous imaging approac hesknown to the skill ed worker, such as non-invasive in vivo imaging techniques commonly used in the medica arenal for, example: compute tomographyd imaging (CT scannin opticag), l imaging (including direct fluores, cent, and bioluminescent imaging), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), ultrasound, and x-ra computedy tomography imaging.
VIII. Production or Manufactur of Pharmaceute and/orical Diagnostic Compositions Comprising Cell-Target Molecing ules of the Present Invention 676. 676. 676. id="p-676" id="p-676" id="p-676" id="p-676" id="p-676" id="p-676" id="p-676" id="p-676" id="p-676" id="p-676"
[676] Pharmaceutic accallyepta saltble ors solvates of any of the Shiga toxin effector polypeptides and cell-targeti moleculeng ofs the present invention are within the scope of the present invention. 677. 677. 677. id="p-677" id="p-677" id="p-677" id="p-677" id="p-677" id="p-677" id="p-677" id="p-677" id="p-677" id="p-677"
[677] The ter m"solvate" in the context of the prese ntinvention refe rsto a complex of defined stoichiom etryforme betweend a solute (in casu, a proteinace ous compound or pharmaceutic acceptaally salble thert eof accordi tong the invention) and a solvent. The solven in tthis connection may, for example, be water, ethanol or another pharmaceutic accallyepta ble,typical small-molecly orgaularnic species , such as, but not limite to,d acetic acid or lact acid.ic When the solven in tquestion is water, such a solva iste normally referr toed as a hydrate. 678. 678. 678. id="p-678" id="p-678" id="p-678" id="p-678" id="p-678" id="p-678" id="p-678" id="p-678" id="p-678" id="p-678"
[678] Polypeptide ands protei nsof the prese ntinvention, or salt thereofs may, be formulate as pharmad ceut compical ositions prepared for storage or administration, which typica llycomprise a therapeuticall effectivey amount of a molecule of the present invention, or a sal theret of,in a pharmaceutic acceptaally carrierble The. term "pharmaceuticall acceptay carble rier" includ esany of the standard pharmaceuti carcalrie rs.Pharmaceutic accallyepta carrble iers for therapeut ic molecule use are wel knownl in tire pharmaceutica art, andl are described, for example, in Remington’s Pharmaceutical Sciences (Mack Publishing Co. (A.
Gennaro, ed., 1985). As used herein, "pharmaceutically accepta carrible " erincludes any and all physiologic acceptable,ally t e. compatible, solvents, dispersion media, WO 2016/196344 PCT/US2016/034778 247 coatings anti, microbial agents, isotonic, and absorption delaying agents, and the like.
Pharmaceutically accepta earble ners or diluents include those used in formulations suitable for oral, rect al,nasal or parenter (includial ngsubcutaneous, intramuscular, intravenous intr, ader maland trans, derm administal) ration. Exempla ry' pharmaceutically accepta carrble iers include steril aqueouse solutions or dispersions and ster ilepowders for the extemporaneous preparation of sterile injectable solutions or dispersions. Example ofs suitable aqueous and nonaqueous carriers that may be employed in the pharmaceut compical ositions of the invention includ e water, ethanol, polyols (such as glycero propylenel, glycol, polyethyle glycne ol, and the like) and, suitable mixture thers eof, vegetable oils, such as olive oil, and injectable organic esters such, as ethylole ate.Proper fluidi tycan be maintained, for example, by die use of coating materials such, as lecithin by ,the maintenance of the require particd sizlee in the case of dispersions, and by the use of surfactant In s. certain embodiments, the carri iser suitable for intravenous intra, muscular, subcutaneous, parenteral spinal, or epidermal administration (e.g. by injecti onor infusion). Depending on selected route of administrati theon, protein or other pharmaceuti compcal onen mayt be coated in a materia intel nded to protect the compound from the action of low pH and othe naturalr inactiva tingconditions to which die active protein may encounter when administer toed a patient by a particula router of administration. 679. 679. 679. id="p-679" id="p-679" id="p-679" id="p-679" id="p-679" id="p-679" id="p-679" id="p-679" id="p-679" id="p-679"
[679] 'The formulations of the pharmaceut compical ositions of the invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. In such form the, composition is divide intod unit doses containing appropriate quantitie of sthe active component.
The unit dosage form can be a. packaged preparati theon, package containing discrete quantitie of sthe preparations, for example, packeted tablet capsuless, and, powders m vials or ampoule s.The unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropri atenumber of any of these packaged forms. It may be provided in single dose injecta bleform, for example in the form of a. pen.
Compositions may be formulated for any suitable route and means of administra tion.
Subcutaneous or transderma modesl of administration may be particula suitrlyable for therapeutic proteins described herein. 680. 680. 680. id="p-680" id="p-680" id="p-680" id="p-680" id="p-680" id="p-680" id="p-680" id="p-680" id="p-680" id="p-680"
[680] The pharmaceutica compol sitions of the prese ntinvention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersingagents Prevent. ingthe presence of microorgani maysms be ensured both by sterilizat proceduresion and, by the inclusion of various antibacte andria antil fungal agents, for exampl paraben,e, chlorobutanol phenol,, sorbic acid, and the like.
Isotonic agents such, as sugars, sodium chlori de,and the lik einto the compositions, may also be desirabl Ine. addition, prolonged absorpti ofon the injectable pharmaceutic formal may be brought about by the inclusion of agent whics hdela y absorpti suchon as, aluminum monostearate and gelatin. 681. 681. 681. id="p-681" id="p-681" id="p-681" id="p-681" id="p-681" id="p-681" id="p-681" id="p-681" id="p-681" id="p-681"
[681] A pharmaceuti composical tion of the present inventio alson optiona lly includes a pharmaceutical acceplytable antioxidant Exemplar. pharmacy eutically acceptable antioxidants are wat ersoluble antioxidants such as ascor bicacid, cystei ne hydrochloride, sodium bisulfate sodium, metabisulfit sodiume, sulfi teand the like; oil-solubl antioxie dants, such as ascorbyl palmitat butyle, ated hydroxyani sole (BHA), butylate hydroxytoluened (BHT), lecithin, propylgall alpha-tate, ocopherol, and the like; and metal chelati agents,ng such as citric acid, ethylenediamine tetraacet aciic d(EDTA), sorbit ol,tartaric acid, phosphoric acid, and the like. 682. 682. 682. id="p-682" id="p-682" id="p-682" id="p-682" id="p-682" id="p-682" id="p-682" id="p-682" id="p-682" id="p-682"
[682] In another aspec thet, present invention provides pharmaceuti cal compositions comprising one or a combinat ionof different polypepti desand/or cel­ l target moling ecul ofes the invention, or an ester salt, or amide of any of the foregoing, and at leas onet pharmaceuti calaccelyptabl carriee r. 683. 683. 683. id="p-683" id="p-683" id="p-683" id="p-683" id="p-683" id="p-683" id="p-683" id="p-683" id="p-683" id="p-683"
[683] . Therapeuti composic ti onsare typica llsteyril ande stabl undere the conditions of manufactur ande storage. The compositi mayon be formulat ased a solutio n, microemulsion, liposom ore, other order structureed suitable to high drug concentration. The earner may be a solvent or dispersion medium containing, for exampl wate, er, alcohol such as ethanol, polyol (e.g., glycerol propylene, glyco andl, liqui dpolyethylene glycol), or any suitabl mixte ures. The proper fluidi tymay be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required partic sizele in the case of dispersion and by use of surfactants accord ingto formulation chemistry wel knownl in the art In. certa embodiin ments , isotonic agents e.g.,, sugars and polyalcohols such as mannitol sorbit, ol,or sodium chlori de,may be desirable in the composition. Prolonged absorpt ionof injectable compositi onsmay be brought about by including in the compositi anon agent that delays absorpti foron example, monostearate salt ands gelatin. 684. 684. 684. id="p-684" id="p-684" id="p-684" id="p-684" id="p-684" id="p-684" id="p-684" id="p-684" id="p-684" id="p-684"
[684] Solutions or suspensions used for intradermal or subcutaneous applicati on typical inclly ude one or more of: a ster ilediluent such as wate forr injection, saline -248-WO 2016/196344 PCT/US2016/034778 249 solution, fixed oils, polyethyle glycols,ne glyceri propylenene, glycol or other synthetic solvents; antibacte agentsrial such as benzyl alcohol or methyl parabens ; antioxida suchnts as ascorbic acid or sodium bisulfite; chelatin agentsg such as ethylenediaminete traaacid; buffceticers such as acetates citr, ates or phosphates and; tonici adjuty sting agents such as, e.g., sodium chlor ideor dextrose. The pH can be adjusted with acids or bases such, as hydrochlor acidic or sodium hydroxide, or buffers with citra te,phosphate, acetate and the like. Such preparation mays be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. 685. 685. 685. id="p-685" id="p-685" id="p-685" id="p-685" id="p-685" id="p-685" id="p-685" id="p-685" id="p-685" id="p-685"
[685] Sterile injectable solutions may be prepared by incorporati a polypeptng ide or cell-target moleingcule of the invention in the require amountd in an appropriat e solvent with one or a combinati ofon ingredients describe above,d as require d, follow byed steriliza tionmicrofiltrati Dison.persions may be prepared by incorpora theting active compound into a steril vehiclee that conta insa dispersion medium and other ingredie nts,such as those described above. In the cas eof ster ile powde rsfor the preparati ofon ster ileinjectable solutions, the methods of preparat areion vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredie innt addition to any additional desired ingredient from a sterile-fil tersolutioned thereof. 686. 686. 686. id="p-686" id="p-686" id="p-686" id="p-686" id="p-686" id="p-686" id="p-686" id="p-686" id="p-686" id="p-686"
[686] When a therapeutically effective amount of a polypeptide and/or cell- targeting molecule of the invention is designed to be administered by, e.g, intravenous cutan, eous or subcutaneous injection, the bindin gagent will be in the form of a pyrogen-fr pareee, ntera acceptally aqueousble solution. Methods for preparing parentera accllyepta proteinble solutions, taking into consideratio n appropriate pH, isotonic ity,stability, and the like, are within the skill in the art. A prefer redpharmaceutica composl ition for intravenous, cutaneous or ,subcutaneous injecti onwill contain, in additi onto bindin gagents, an isotonic vehicle such as sodium chlor ideinjection, Ringer injection,'s dextrose injection, dextrose and sodium chlor ideinjection, lacta tedRinger's injectio orn, othe vehicler as known in tire art. A pharmaceutica composil tion of the present invention may also conta in stabilize presers, rvatives buffe, rs, antioxidants or other, additives wel knownl to those of skill in the art. 687. 687. 687. id="p-687" id="p-687" id="p-687" id="p-687" id="p-687" id="p-687" id="p-687" id="p-687" id="p-687" id="p-687"
[687] As describe elsewherd herein,e a polypeptide and/or cell-target moleculeing of the prese ntinvention may be prepared with carrie thatrs will prote thect active WO 2016/196344 PCT/US2016/034778 250 therapeutic agent against rapid releas suche, as a controll releedase formulation, including implants, transdermal patche ands, microencapsulate deliverd systey ms.
Biodegradable, biocompatibl polymerse can be used, such as ethylene vinyl acetate , polyanhydrides polyglycolic, acid collagen,, polyorthoeste and polylacticrs, acid .
Many methods for the preparat ofion such formulations are patented or generall y known to those skilled in the art (see e.g. Sustained and Controlled Release Drug Delivery Systems (Robinson J, ed., Marcel Dekker, Inc., NY, U.S., 1978)). 688. 688. 688. id="p-688" id="p-688" id="p-688" id="p-688" id="p-688" id="p-688" id="p-688" id="p-688" id="p-688" id="p-688"
[688] In certa embodimein nts, the compositi ofon the prese ntinvention (e.g. a pharmaceuti and/orcal diagnosti compoc siti on)may be formulated to ensure a desired in vivo distribution of a cell-targeti molengcule of the present inventi on.For example, the blood-br ainbarri excludeer manys large and/o hydrophilicr compounds To. targ aet therapeutic molecule or composition of the prese nt invention to a particular m vivo location, they can be formulate ford, example, in liposomes which may comprise one or more moietie thats are selective tralynspo rted into specif iccells or organs, thus enhancing targeted drag delivery. Exemplar7 y targeti moietng ies include folate or biotin; mannosid antibodies;es; surfactant protein A recepto pl20r; catenin and the like. 689. 689. 689. id="p-689" id="p-689" id="p-689" id="p-689" id="p-689" id="p-689" id="p-689" id="p-689" id="p-689" id="p-689"
[689] Pharmaceutic composital ions include parente formularal tions designed to be used as implants or particul systemsate Example. ofs implants are depo t formulations composed of polymeric or hydrophobic compone ntssuch as emulsions, ion exchange resins, and soluble salt solutions. Examples of particulat sysetems are microspheres, micropartic nanocles, apsu nanospherles, andes nanopartic, (seeles e.g, Honda M^ et al., Int J Nanomedicine 8: 495-503 (2013); Shanna A et al., Biomed Res Int2013: 960821 (2013); Ramishetti S, Huang L, Ther Deliv 3: 1429-45 (2012)).
Controlled release formulations may be prepared using polyme sensirs tive to ions , such as, e.g. liposomes polaxatner, 407, and hydroxyapatite.
IX. Polynucleoti Expressdes. ion Vectors, and Host Cell ofs the Prese ntInvention 690. 690. 690. id="p-690" id="p-690" id="p-690" id="p-690" id="p-690" id="p-690" id="p-690" id="p-690" id="p-690" id="p-690"
[690] Beyond the polypeptides and cell-targetin molecg ules of the present invention, the polynucleoti thatdes encode the polypeptides and cell-targeting molecules of the invention, or functional portions there of,are also encompas sed within the scope of the present invention. The ter m"polynucleoti is de"equivalent to the ter m"nuclei acid,c " each of whic hincludes one or more of; polymers of deoxyribonucleic acid (DNAs s), polymers of ribonucl eicacid (RNAss ), analogs ofWO 2016/196344 PCT/US2016/034778 251 these DNAs or RNAs generat usinged nucleotide analogs, and derivative s, fragmen andts homologs thereof. The polynucleotide of the present invention may be single-, double or-, triple-stra nded.Such polynucleoti aredes specifica lly disclosed to include all polynucleoti capdesable of encoding an exemplar proteiy' n, for example, taking into account the wobble known to be tolerated in the thir d position of RNA codons, yet encoding for the same ammo acid as a differe RNAnt codon (see Stothard P, Biotechmqnes 28: 1102-4 (2000)). 691. 691. 691. id="p-691" id="p-691" id="p-691" id="p-691" id="p-691" id="p-691" id="p-691" id="p-691" id="p-691" id="p-691"
[691] In one aspect, the prese ntinvention provides polynucleotides which encode a Shiga toxin effector polypeptide and/or cell-targetin molegcule of the present invention, or a fragment or derivative thereof. The polynucleoti maydes include, e.g., a nucle icacid sequence encoding a polypept ideat leas 50%,t 55% , 60% , 65%, 70%, 15%, 80%, 85%, 90%, 95%, 99% or more, identic toal a polypeptide comprising one of the amino acid sequenc ofes a polypept ideor cell-targeting molecule of the present invention. The invention als oincludes polynucleoti des comprising nucleoti sedequences that hybridi zeunder string entconditions to a polynucleotid whiche encodes Shiga toxin effector polypeptide and/o cell-tar rgeting molecule of the invention, or a fragment or derivative thereof, or the antisens or e complement of any such sequence. 692. 692. 692. id="p-692" id="p-692" id="p-692" id="p-692" id="p-692" id="p-692" id="p-692" id="p-692" id="p-692" id="p-692"
[692] Derivati vesor analo ofgs the molecules of the present invention (e.g., Shiga toxin effector polypepti desof the prese ntinvention and cell-targeti moleculeng s comprising the same) include inter, alia, polynucleot (oride polypeptide molecul) es having regions that are substantiall homoloy gous to the polynucleotides (or Shiga, toxin effector polypepti desand cell-targeti molecng ules of the prese ntinvention ), e.g. by at least abou t45%, 50%, 70%, 80%, 95%, 98%, or even 99% identity (with a prefer redidenti ofty 80-99%) over a polynucleotid (or polypeptide)e sequence of the same size or when compared to an aligne sequed nce in which the alignme isnt done by a computer homology program known in the art. An exemplar programy' is the GAP program (Wisconsin Sequence Analys Pacis kage Vers, ion 8 for UNIX, Genetics Computer Group, Univers ityResearc Park,h Madison, WI, U.S.) using the default setting whichs, uses the algorit ofhm Smith T, Waterma M,n Adv Appl Math 2: 482-9 (1981). Also includ edare polynucleoti capadesble of hybridizing to tire complement of a sequence encoding the cell-targetin proteg ins of the invention under stringent conditions (see e.g. Ausubel F et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York, NY, U.S., 1993)), and below. Stringe ntWO 2016/196344 PCT/US2016/034778 252 conditions are known to those skilled in the art and may be found, e.g., in Current Protocols in Molecular Biology (John Wiley & Sons, NY, U.S., Ch. Sec. 6.3.1-6.3.6 (1989)). 693. 693. 693. id="p-693" id="p-693" id="p-693" id="p-693" id="p-693" id="p-693" id="p-693" id="p-693" id="p-693" id="p-693"
[693] The present invention furthe providesr expression vectors that comprise the polynucleoti withindes the scope of the present invention. The polynucleoti des capable of encoding the Shiga toxin effector polypeptide and/ors cell-targeting molecules of the invention may be inserted into known vectors including, bacteri al plasmids, vira vectl ors and phage vectors using, material and methods well known in the art to produc expresse ionvectors. Such express ionvectors will include the polynucleoti necessdes ary to suppor productiont of contempla Shigated toxin effector polypeptides and/or cell-targeti molecng ules of the invention within any host cell of choic ore cell-fre expre ess ionsystems (e.g. pTxbl and pIVEX2.3). The specif icpolynucleoti comprdes isin exprg ess ionvectors for use with specif ictypes of host cells or cell-f reeexpress ionsystems are wel knownl to one of ordinar skilly in the art, can be determined using routine experimenta and/ortion, may be purchased. 694. 694. 694. id="p-694" id="p-694" id="p-694" id="p-694" id="p-694" id="p-694" id="p-694" id="p-694" id="p-694" id="p-694"
[694] The term "express ionvector," as used herein, refe rsto a polynucleot ide, linea orr circul ar,comprising one or more express ionunits. The ter m"express ion unit" denotes a polynucleot segmentide encoding a polypeptide of interest and capable of providing expression of the nucleic acid segment in a host cell An. express ionunit typically compris aes transcriptio promon ter an open, reading frame encoding the polypeptide of interest, and a transcription terminato all r,in operabl e configuration. An expression vector conta insone or more express ionunits. Tirus, in tire context of the prese ntinvention, an express ionvector encoding a Shiga toxin effector polypeptide and/or cell-target moleingcule of the invention comprisin a g single polypeptide chain includes at least an express ionunit for the singl e polypept idechain, where aas protei comprising,n e.g. two or more polypeptide chains (e.g. one chai comprisingn a V1. domai andn a second chain comprising a Vh domai linkedn to a toxin effector polypeptide incl) udes at leas twot express ionunits, one for eac hof the two polypeptide chains of the protei n.For expression of multi- chain cell-targetin proteinsg of the invention, an express ionunit for each polypeptide chain may als obe separat contaely ined on different express ionvectors (e.g. expression may be achieved wit ha singl hoste cel intol which express ion vectors for each polypeptide chain has been introduced).WO 2016/196344 PCT/US2016/034778 253 695. 695. 695. id="p-695" id="p-695" id="p-695" id="p-695" id="p-695" id="p-695" id="p-695" id="p-695" id="p-695" id="p-695"
[695] Expression vectors capable of directin transg ient or stabl expresse ionof polypepti desand protei nsare wel knownl in the art. The express ionvectors generally include but, are not limite to,d one or more of the followi ng:a heterologous signal sequence or peptide, an origin of replication, one or more marke genes,r an enhancer element, a promoter and, a transcription termination sequence eac, hof which is wel knownl in the art. Optional regulatory7 contr ol sequence integras, tionsequence ands, useful markers that can be employed are known in the art. 696. 696. 696. id="p-696" id="p-696" id="p-696" id="p-696" id="p-696" id="p-696" id="p-696" id="p-696" id="p-696" id="p-696"
[696] Ilie term "host cell" refe rsto a cell which can suppor thet replicatio or n express ionof the express ionvector Host. cells may be prokaryotic cells such, as E. colt or eukaryoti cellsc (e.g. yeast, insect, amphibian, bird, or mammalia cellsn ).
Creation and isolation of host cell lines comprisin a gpolynucleot ofide the invention or capable of produci nga polypeptide and/or cell-targetin molegcule of the present invention can be accomplis hedusing standa technird ques known in the art. 697. 697. 697. id="p-697" id="p-697" id="p-697" id="p-697" id="p-697" id="p-697" id="p-697" id="p-697" id="p-697" id="p-697"
[697] Shiga toxin effecto polypepr tides and/or proteins within the scope of the present invention may be variant or sderivati ofves the polypeptides and molecules described herei thatn are produc edby modifying the polynucleot encideoding a polypept ideand/or proteinaceous component of a. cell-targeti molengcule by altering one or more amino acid ors deleting or inserting one or more amino acids that may render it more suitable to achieve desire propertd iessuch, as more optimal express ionby a host cell.
X. Molecules of the Present Invention Immobilized on Solid Substrates 698. 698. 698. id="p-698" id="p-698" id="p-698" id="p-698" id="p-698" id="p-698" id="p-698" id="p-698" id="p-698" id="p-698"
[698] Certain embodimen ofts the prese ntinvention include a molecule of the present invention (e.g. a Shiga toxin effecto polypr epti ade, cell-targeti molecng ule, fusion protein, or polynucleot ofide the prese ntinvention), or any effecto fragmr ent thereof, immobilized on a solid substrate Solid. substrates contemplat hereed in include but, are not limited, to, microbeads nanoparti, cles,polyme rs,matr ix materia microarraysls, micro, tit plater es, or any solid surface known in the art (see e.g. US 7,771,955). In accordance with thes embodimee nts, a molecule of the present invention may be covalently or non-covale linkedndy to a soli dsubstrat e, such as, e.g., a. bead par, tic orle, plate, using techniques known to the skilled worker (see e.g. Jung Y et al., Analyst 133: 697-701 (2008)). Immobilized molecules of the invention may7 be used for screening applications using techniques known m the artWO 2016/196344 PCT/US2016/034778 254 (see e.g. Bradbury A et al., Nat Biolechnol 29: 245-54 (2011); Sutton C, BrJ Pharmacol 166: 457-75 (2012): Diamant Le et al., Protein Eng Des Sei 26: 713-24 (2013): Houlihan G et al., J Immunol Methods 405: 47-56 (2014)). 699. 699. 699. id="p-699" id="p-699" id="p-699" id="p-699" id="p-699" id="p-699" id="p-699" id="p-699" id="p-699" id="p-699"
[699] Non-limiti examng ples of solid substrate to whichs a molecule of the invention may be immobilized on include: microbeads nanopa, rticles polymers,, nanopolym nanotubeers, magnetics, beads, paramagnetic beads super, paramagnet ic beads, streptavidi coatedn beads, reverse-phas magne etic beads car, boxy terminated beads, hydrazine terminate beads,d silica (sodium silic a)beads and iminodiace tic acid (IDA) -modifie beads,d aldehyde-modif beads,ied epoxy-activa beads,ted diaminodipropylami (DADPneA) -modified beads (beads wit hprimary amine surfa cegroup), biodegrad ablepolymeric beads, polystyrene substrate amino-s, polystyre partine cles carboxyl-polys, partityrenecles epoxy-polystyr, pareneticles , dimethylamino-polys partictyreneles, hydroxy-polyst partiyrenecles colo, red particles flow, cytometr partiy' cles sulf, onate-polys partityrenecles nitrocellulose, surfac es,reinforce nitrocd ell ulosemembrane nylons, membrane glasss, surfaces , activat glassed surfac esactivat, quarted surz fac espolyvinyl, idene difluoride (PVDF) membrane polyacrylas, mide-bas substrateed poly-vis, nyl chlor idesubstrate poly-s, methy methal cryla substrate tes, poly(dimethyl siloxane) substrate ands, photopolymers which contain photoreac spectiveies (such as nitrene carbeness, and, ketyl radica capals) ble of forming covalent linkages Other. examples of soli d substra testo which a molecule of the invention may' be immobilized on are commonly used in molecular display systems such, as, e.g., cellular surfaces, phages and, virus particles.
XL Delive־ry Devices and Kits 700. 700. 700. id="p-700" id="p-700" id="p-700" id="p-700" id="p-700" id="p-700" id="p-700" id="p-700" id="p-700" id="p-700"
[700] In certa emboin diments, the invention relates to a device comprisi ngone or more compositions of matte ofr the present invention, such as a pharmaceuti cal composition or diagnost compositic ion, for delivery to a subject in need thereof.
Thus, a delivery devic compre isi ngone or more compositions of the present invention can be used to administer to a patient a composition of matt ofer the present invention by various delivery methods, including: intravenous , subcutaneous, intramuscular or intraperitone injealctio oraln; administra tion; transderma admil nistrati pulmonaryon; or transmucos administraal tion;WO 2016/196344 PCT/US2016/034778 255 administration by implant, osmoti pump,c cartri dgeor micro pump; or by other means recogniz byed a person of skil inl the art. 701. 701. 701. id="p-701" id="p-701" id="p-701" id="p-701" id="p-701" id="p-701" id="p-701" id="p-701" id="p-701" id="p-701"
[701] Also withi then scope of the prese ntinvention are kits comprising at leas t one composition of matte ofr the invention, and optional packaly, ging and instructions for use. Kits may be useful for drug administration and/or diagnos tic informati gathering.on A kit of the invention may optionally comprise at leas onet additional reagent (e.g., standards mar, kers and the like). Kits typically includ ae labe indical ting the intended use of the contents of the kit. The kit may further comprise reagents and othe toolsr for detecting a cell type (e.g. a tumor cell) in a sample or in a subject, or for diagnos ingwhether a patient belongs to a group that responds to a. therapeuti strac tegy whic hmakes use of a. compound, composition, or related method of the prese ntinvention, e.g.. such as a method describe hered in.
XII. Methods for Using Cell-Targe Moleculting ofes the Present Inventi onand/or Pharmaceutical and/o Diagnosr ticCompositions Thereof 702. 702. 702. id="p-702" id="p-702" id="p-702" id="p-702" id="p-702" id="p-702" id="p-702" id="p-702" id="p-702" id="p-702"
[702] Generally, it is an object of the present invention to provide pharmacologica activelly agents, as wel asl compositions comprisin theg same, that can be used in the prevention and/or treatme ofnt disease disors, ders and, conditions, such as certain cancer tumorss, growth, abnormalit immuneies, disorders or furthe, r pathological conditions mentione hereid n.Accordingly, the present invention provide methods ofs using the polypeptide cell-s, target molecules,ing and pharmaceutica compol sitions of the invention for the targeted killing of cell fors, delivering additional exogenou materias intols targeted cells for, labeling of the interiors of targete celdls, for collect diagnostiing informc ation, for the deliver ingof T-cell epitopes to the MHC class I presentati pathwayon of targe cellt ands, for treating diseases, disorde andrs, conditions as described herein. For example, the methods of the prese ntinvention may be used to prevent or treat cance rs,canc er initiation, tumor initiation, metasta and/orsis, diseas reoce currence. 703. 703. 703. id="p-703" id="p-703" id="p-703" id="p-703" id="p-703" id="p-703" id="p-703" id="p-703" id="p-703" id="p-703"
[703] In particula it isr, an object of the invention to provide such pharmacologica activelly agents, compositions, and/o methodsr that have certain advantages compared to the agents, compositions and/or, methods that are currently known in the art. According thely, prese ntinvention provides methods of using Shiga toxin effector polypepti desand cell-targeti molecng ules with specified protei n sequenc andes pharmaceutica compol sitions thereof. For exampl anye, of the amino WO 2016/196344 PCT/US2016/034778 256 acid sequences in SEQ ID NOs: 6-354 and 370-513 may be specificall utilizey asd a component of the cell-targeting molecule used in the followi methodsng or any method for using a cell-target moleingcule known to the skill edworke suchr, as, e.g., various methods described in WO 2014/164680, WO 2014/164693, WO 2015/138435, WO 2015/138452, WO 2015/113005, WO 2015/113007, WO 2015/191764, US20150259428, 62/168,758, 62/168,759, 62/168,760, 62/168,761, 62/168,762, 62/168,763, and PCT/US2016/016580. 704. 704. 704. id="p-704" id="p-704" id="p-704" id="p-704" id="p-704" id="p-704" id="p-704" id="p-704" id="p-704" id="p-704"
[704] The present invention provides methods of killing a ceil comprisin theg ste p of contact theing cell either, in vitro or tn vivo, with a Shiga toxin effector polypepti cell-de, target moleingcule or ,pharmaceutica composil tion of the present invention. The Shiga toxin effector polypeptide cell-s, target moleingcule ands, pharmaceuti compocal sitions of the prese ntinvention can be used to kill a specifi c cell type upon contact aing cell or cells wit hone of the claimed compositi onsof matter. In certain embodiment a s,cell-targetin molecg ule or pharmaceutic al composition of the present invention can be used to kill specif iccel typesl in a mixtur ofe different cel types,l such as mixtur escomprisin canceg cellr infes, cte d cells, and/or hematologica cell ls.In certai embodimen nts, a cell-target molecing ule, or pharmaceut composiical tion of the present invention can be used to kill canc er cel lsin a mixture of different cell types In. certai embodn iment a cytotoxics, Shiga cell-targetin molecg ule, or pharmaceutic composial tion of the present invention can be used to kill specif iccell types in a mixture of differ entcel typesl such, as pre- transplantation tissue s.In certa embodimein nts, a Shiga, toxin effector polypeptide, cell-targetin molecg ule, or pharmaceutic composal ition of the present invention can be used to kill specif iccell types in a mixture of cell types such, as pre- administrati tisonsue material for therapeuti purposes.c In certain embodiments, a cell-targetin molegcule or pharmaceutic composal ition of the prese ntinvention can be used to selective killly cells infecte byd viruses or microorganism or others, wise selective killly cells expressing a particular extracel targetlular biomolecule, such as a cell surface biomolecule. The Shiga, toxin effector polypeptide cell-tas, rgeting molecule ands, pharmaceuti compcal ositions of the present invention have varie d applications including,, e.g., uses in depleti unwantedng cell types from tissues eithe inr vitro or in vivo, uses in modulat ingimmune response to streat graft versus host use, s as antivir agenal ts, uses as anti-para sitiagenc ts, and uses in purging transplantation tissues of unwanted cell types.WO 2016/196344 PCT/US2016/034778 257 705. 705. 705. id="p-705" id="p-705" id="p-705" id="p-705" id="p-705" id="p-705" id="p-705" id="p-705" id="p-705" id="p-705"
[705] In certa emboin diments, certain Shiga toxin effector polypeptide ceil-s, targeting molecule ands, pharmaceuti compositical onsof the prese ntinvention, alone or in combinat ionwith other compounds or pharmaceut compositions,ical can show potent cell-kill activity when administe tored a. population of cells in, vitro or in vivo in a subject such as in a patient in need of treatment. By targeti theng delive׳ r}of enzymatically active Shiga toxin A Subunit effecto polypeptidesr and/or T-cell epitopes using high-affinity binding regions to specif iccell types, cell-kill activitie cans be restricted to specificall andy selective killingly certa ceilin types within an organism, such as certa cancein celr ls, neoplas celticls, malignant cells , non-malignant tumor cell and/os, infecr ted cells. 706. 706. 706. id="p-706" id="p-706" id="p-706" id="p-706" id="p-706" id="p-706" id="p-706" id="p-706" id="p-706" id="p-706"
[706] The present invention provide a smethod of killing a cell in a patient in need thereof, the method comprisi ngthe step of administerin to theg patient at least one cell-target moleculeing of the present invention or a pharmaceut composiical tion thereof. 707. 707. 707. id="p-707" id="p-707" id="p-707" id="p-707" id="p-707" id="p-707" id="p-707" id="p-707" id="p-707" id="p-707"
[707] In certa emboin diments, the ceil-target moleingcule of the present invention or pharmaceut compoical sitions there canof be used to kill a cance ceir lin a patient by targeting an extracellular biomolec ulefound physically coupled with a canc eror tumor cell Hie. term "scancer ceil" or "cancerou cells" refe rsto various neoplas tic cells which grow' and divide in an abnorma accllyelera and/oted unregr ulate fasdhion and wall be clear to the skilled person. The term "tumor cell" includes both malignant and non-malignant ceil s.General cancerly, and/os tumorsr can be defined as diseases, disorde orrs, conditions that are amenable to treatment and/or prevention. Hie cancers and tumors (either malignant or non-malignant) which are comprise ofd cance ceilsr and/o tumorr ceils which may benefit from methods and compositions of the invention will be clea tor the skilled person. Neopiasti cellsc are often associated with one or more of the following: unregulated growt lach, ofk differentiation, loca tissul einvasion, angiogenesis and ,metasta sis.Ilie diseases , disorde andrs, conditions resulting from cancer and/os tumorsr (either maligna ornt non-malignant) which may benefit from the methods and compositions of the present invention targeting certain cance cellsr and/or tumor cells will be clear to the skilled person. 708. 708. 708. id="p-708" id="p-708" id="p-708" id="p-708" id="p-708" id="p-708" id="p-708" id="p-708" id="p-708" id="p-708"
[708] Certai embodn imen ofts the cell-target molecing ules and compositions of the present invention may be used to kill cance stemr cells, tumor stem cells, pre- malignant cancer-initi atingcells, and tumor-initiating cells, which commonly are WO 2016/196344 PCT/US2016/034778 258 slow dividing and resistant to cance therar pies like chemother andapy radiation. For example, acute myeloid leukemia (AMLss ) may be treated with the present invention by killing AML stem, cells and/or dormant AML progenitor cells (see e.g.
Shlush L et al., Blood 120: 603-12 (2012)). Cancer stem cells often overexpres cell s surfa cetargets such, as, e.g.. CD44, CD200, and others listed herein, which can be targets of certai bindingn regio nsof certain embodiments of the cell-targetin g molecules of the prese ntinvention (see e.g. Kawasaki B et al, Biochem Biophys Res Commun 364:778-82 (2007): Reim F et al., CancerRes 69: 8058-66 (2009)). 709. 709. 709. id="p-709" id="p-709" id="p-709" id="p-709" id="p-709" id="p-709" id="p-709" id="p-709" id="p-709" id="p-709"
[709] Because of the Shiga toxin A Subuni tbased mechanis ofm action, compositions of matte ofr the present invention may be more effectivel usedy in methods involving thei combinar tion with or, in complementary fashion with other therapies such, as, e.g., chemotherap immiesunothe, rapi radiaes, tion, stem cei l transplantat andion, immune checkpoint inhibitors and/o, effer ctive against chemoresistant/radiation-re and/o sisresr tantting tumor cells/tumor initiating cells/ste cellm s.Similar ly,compositions of matter of the present invention may be more effectively used in methods involving in combination with othe celr l-target ed therapies targeting othe thanr the same epitope on, non-overlapping, or differ ent target fors the same diseas disore der or condition. 710. 710. 710. id="p-710" id="p-710" id="p-710" id="p-710" id="p-710" id="p-710" id="p-710" id="p-710" id="p-710" id="p-710"
[710] Certa embodin imen ofts the cell-targetin molecg ules of the prese ntinvention, or pharmaceutica compositl ions thereof, can be used to kill an immune cell (whethe r healthy or malignant) in a patient by targeting an extracel biomoleclular ulefound physically coupled with an immune cell. 711. 711. 711. id="p-711" id="p-711" id="p-711" id="p-711" id="p-711" id="p-711" id="p-711" id="p-711" id="p-711" id="p-711"
[711] It is withi then scope of the prese ntinvention to utiliz ae cell-targeting molecule of the prese ntinvention, or pharmaceuti composical tion there of,for the purposes of purging patient cel populationsl (e.g. bone marrow) of malignant, neoplast oric, otherwis unwantede T-cells and/or B-cell ands then reinfusing the T- cell and/or B-cells depleted material into the patient (see e.g. van Heeckere Wn et al., Br J Haematol 132: 42-55 (2006); (see e.g. Alpdog anO, van den Brink M, Semin Oncol 39: 629-42 (2012)). 712. 712. 712. id="p-712" id="p-712" id="p-712" id="p-712" id="p-712" id="p-712" id="p-712" id="p-712" id="p-712" id="p-712"
[712] It is withi then scope of the prese ntinvention to utiliz thee cell-targetin g molecule of the prese ntinvention, or pharmaceuti composical tion there of,for the purposes of ex vivo deplet ionof T cel lsand/o B-cer lls from isolated cell populations removed from a patient. In one non-limiti example,ng the cell-targetin molegcule of the invention can be used in a metho ford prophylaxis of organ and/or tissue WO 2016/196344 PCT/US2016/034778 259 transpla rejentction wherei then donor organ or tissue is perfuse priord to transplant wit ha cytotoxic, cell-target molecing ule of the invention or a pharmaceuti cal composition thereof in order to purge the organ of donor T-cells and/o B-cellsr (see e.g. Alpdogan O, van den Brink M, Semin Oncol 39: 629-42 (2012)). 713. 713. 713. id="p-713" id="p-713" id="p-713" id="p-713" id="p-713" id="p-713" id="p-713" id="p-713" id="p-713" id="p-713"
[713] It is als withino the scope of the prese ntinvention to utiliz ethe cell-targetin g molecule of the invention, or pharmaceut composiical tion there of,for the purpos es of depleting T-cells and/or B-cells from a donor cell population as a prophylaxis against graft-versus disea-hostse, and induction of tolerance, in a patient to undergo a bone marrow and or stem cell transpla (seent e.g. van Heeckere Wn et al., Br J Haematol 132: 42-55 (2006); (see e.g. Alpdog anO, van den Brink M, Semin Oncol 39: 629-42 (2012)). 714. 714. 714. id="p-714" id="p-714" id="p-714" id="p-714" id="p-714" id="p-714" id="p-714" id="p-714" id="p-714" id="p-714"
[714] In certa embodimein nts of the Shiga toxin effecto polypeptider or cell - targeting molecule of the present invention, or pharmaceutic compoal sitions thereof , can be used to kill an infecte celld in a patient by targeting an extracellula r biomolecul founde physically coupled with an infecte celd l. 715. 715. 715. id="p-715" id="p-715" id="p-715" id="p-715" id="p-715" id="p-715" id="p-715" id="p-715" id="p-715" id="p-715"
[715] In certa embodimein nts of the cell-targeti moleculeng ofs the present invention, or pharmaceut compical ositions there of,can be used to "seed" a locu s within a chordate with non-self T-ce, ll epitope-pept presentingide cells in order to activat thee immune system to enhance policing of the locu s.In certain further embodiments of this "seeding" method of the prese ntinvention, the locus is a tumor mass or infecte tissud esite. In preferr emboded imen ofts this "seeding" method of the prese ntinvention, the non-self, T-cell epitope-pept iside selected from the group consisting of: peptides not already presented by the target ceils of the cell-targetin g molecule peptides, not present within any protei expren ss byed the targe cellt , peptides not prese ntwithin the proteo meor transcriptome of the targe celt l,peptides not present in the extracellu microelar nvironme of thent site to be seeded, and peptides not prese ntm the tumor mass or infect tissue site to be targeting. 716. 716. 716. id="p-716" id="p-716" id="p-716" id="p-716" id="p-716" id="p-716" id="p-716" id="p-716" id="p-716" id="p-716"
[716] This "seeding" method functions to labe onel or more targe cellst within a chordate wdth one or more MHC class I presented T-cell epitopes for recognition by effector T-ceils and activation of downstr eamimmune responses By. exploiting the cell internaliz intracing, ellular routlying, and T-cell epitope delivering functions of the cell-targeti molecng ules of the prese ntinvention, the targe cellst w'hich display the delivered T-cell epitope are harnes sedto induc erecognition of the presenting targe cetll by host T-cells and induction of furthe immuner responses including WO 2016/196344 PCT/US2016/034778 260 target-cell-killing by CTLs. Tliis "seeding" method of using a cell-targeting molecule of the prese ntinvention can provide a temporar vaccination-efy by fect inducing adaptive immune response to satta theck cells within the seeded microenvironment, such as, e.g. a tumor mass or infecte tissd ue site, whether presenting a cell-targetin molecg ule-delive T-ceredll epitope( ors) not. This "seeding" method may also induc thee breakin ofg immuno-tolera to ncea targe celt l population, a tumor mass and/or, infected tissu esite withi an chordate. 717. 717. 717. id="p-717" id="p-717" id="p-717" id="p-717" id="p-717" id="p-717" id="p-717" id="p-717" id="p-717" id="p-717"
[717] Certain methods of the prese ntinvention involving the seeding of a locu s within a chordat withe one or more antigenic and/o immunogenir epitc opes may be combined with the administration of immunologic adjuvants, whether administere d loca llyor systemical toly, stimulat thee immune respons toe certain antige ns,such as, e.g., the co-administratio of a composin tion of the prese ntinvention wit hone or more immunologic adjuvants like a cytokine, bacteri produal ct, or plant saponi n.
Other examples of immunologic adjuvant whics hmay be suitable for use in the methods of the present invention include aluminu msalt ands oils, such as, e.g, alums alum, inum hydroxide, miner aloils, squalene paraffin, oils, peanut oils, and thimerosal. 718. 718. 718. id="p-718" id="p-718" id="p-718" id="p-718" id="p-718" id="p-718" id="p-718" id="p-718" id="p-718" id="p-718"
[718] Additiona thelly, prese ntinvention provides a method of treating a disease, disorder, or condition in a patient comprising the step of administerin to ag patient in need there aof therapeuticall effectiy amountve of at leas onet of the cell-targeting molecules of the present invention, or a pharmaceut comica positl ion thereof.
Contempla disetedase disordes, andrs, conditions that can be treated using this method include cance rs,malignant tumors non-malignant, tumors, growth abnormalit immies,une disorde andrs, microbial infections Administ. rati ofon a "therapeutica effectivelly dosage" of a composition of the present inventi oncan resul int a decrea inse severity of diseas symptomse an, increas ine frequency and duration of disease symptom-f reeperiods or, a prevention of impairment or disabilit duey to the diseas affle iction. 719. 719. 719. id="p-719" id="p-719" id="p-719" id="p-719" id="p-719" id="p-719" id="p-719" id="p-719" id="p-719" id="p-719"
[719] The therapeutically effective amount of a composition of the present invention will depend on the route of administrat theion, type of organis beingm treated, and tire physical characteristics of the specifi patientc under considera tion.
These factors and their relationship to determini thisng amount are wel knownl to skilled practition iners the medic alarts This. amount and the method of administration can be tailor toed achieve optimal efficacy, and may depend on such WO 2016/196344 PCT/US2016/034778 261 factors as weight, diet, concurre medicant tion and othe factr ors, wel knownl to those skilled m the medica artsl The. dosage size sand dosing regimen most appropriate for human use may be guide dby the results obtain edby the prese ntinvention, and may be confirmed in properly designe clinicd triaal ls.An effecti dosave ge and treatme protocolnt may be determined by conventional means, starti withng a low' dose in laborator animalsy' and then increasin tireg dosage while monitoring the effects and, systematic varyinally theg dosage regimen as well. Numerous factors may be take inton consideration by a clinicia whenn determin aning optimal dosage for a given subject. Such considerati areons known to the skill edperson. 720. 720. 720. id="p-720" id="p-720" id="p-720" id="p-720" id="p-720" id="p-720" id="p-720" id="p-720" id="p-720" id="p-720"
[720] An accepta routeble of administrat mayion refe tor any administrati on pathway known in the art, including but not limite tod aerosol, enteral nasal, , ophthalmic, oral, parenter rectal,al, vaginal, or transderma (e.g. ltopica l administration of a cream, gel or ointmen ort, by means of a transderm patch)al .
"Parentera adminisl trat" ision typical asslyocia tedwit hinjection at or in communication wit hthe intended site of action including, infraorb italinfusi, on, intraarter intrial,acapsular intraca, rdia intradermac, intral, muscul intraar, peritoneal , intrapulmonary, intraspina intrl, aste mal,intrathecal, intrauteri intravene, nous , subarachnoid, subcapsula subcutaneous,r, transmucos oral, transtrache al administration. 721. 721. 721. id="p-721" id="p-721" id="p-721" id="p-721" id="p-721" id="p-721" id="p-721" id="p-721" id="p-721" id="p-721"
[721] For administration of a pharmaceutic composial tion of the present invention, the dosage range will generally be from about 0.001 to 10 milligra perms kilogra m (mg/kg), and more, usually 0.001 to 0.5 mg/kg, of the subject’s body weight.
Exemplary dosages may be 0.01 mg/kg body weight, 0.03 mg/kg body weight, 0.07 mg/kg body weight, 0.9 mg/kg body weight or 0.1 mg/kg body weight or within the range of 0.01 to 0.1 mg/kg. An exempla treatmentry' regim ise a. once or twic daile y administrati or on,a once or twice weekly administrat onceion, every' two weeks , once ever y'three weeks once, every' four weeks, once a month, once every two or three months or once every' three to 6 months. Dosage mays be selected and readjus tedby the skill edhealth care professional as require tod maximize therapeutic benefit for a particula patirent. 722. 722. 722. id="p-722" id="p-722" id="p-722" id="p-722" id="p-722" id="p-722" id="p-722" id="p-722" id="p-722" id="p-722"
[722] Pharmaceut compoical sitions of the prese ntinvention will typical bely administer to edthe same patient on multiple occasions. Intervals between singl e dosages can be, for example, two to five days, weekl monthly,y, every two or three months ever, sixy months or, yearly. Intervals between administrati canons also be WO 2016/196344 PCT/US2016/034778 262 irregular, based on regulati bloodng levels or othe marr kers in the subject or patient.
Dosage regimens for a composition of the prese ntinvention include intravenous administra oftion 1 mg/kg body weight or 3 mg/kg body weight with the composition administer everyed ־ two to four weeks for six dosage thens, every־ three months at 3 mg/kg body weight or 1 mg/kg body weight. 723. 723. 723. id="p-723" id="p-723" id="p-723" id="p-723" id="p-723" id="p-723" id="p-723" id="p-723" id="p-723" id="p-723"
[723] A pharmaceut compositionical of the prese ntinvention may be administer ed via one or more routes of administrat usingion, one or more of a vari ety of methods known in the art. As will be apprecia byted the skill edworker, the route and/or mode of administration will vary depending upon the desired result Routes. ofs administra fortion cell-target moleculeing ands phannaceutica composl ition s of the present invention include, e.g. intravenous intra, muscular intrader, mal, intraperitoneal subcutaneous,, spina l,or other parentera routesl of administrat forion, example by injection or infusion. For othe embodimer nts, a cell-target moleingcule or phannaceutic composal ition of the invention may be administere by ad non- parentera route,l such as atopical, epiderma orl mucosal route of administrat forion, example, intranasa orallly, ly,vaginally, rectally, sublingually, or topically. 724. 724. 724. id="p-724" id="p-724" id="p-724" id="p-724" id="p-724" id="p-724" id="p-724" id="p-724" id="p-724" id="p-724"
[724] Therapeutic cell-targeti moleculeng ors pharmaceutic compositional s of the present invention may be administered with one or more of a variety of medical devices known in the art. For example, in one embodimen a t,phannaceutica l composition of the invention may be administer withed a needleles hypodermics injection device Example. ofs well-known implants and modules usefi llin the present invention are in the art, including e.g., implantab miclero-infusi pumpson for controll rated delivee ־;ry devices for administerin throughg the skin infus; ion pumps for delivery' at a precise infusion rate: variable flow implantable infusion device fors continuous drug deliver־; andy osmoti drugc deliver־ systey ms. These and other such implan ts,delivery־ systems and, modul esare known to those skilled in the art. 725. 725. 725. id="p-725" id="p-725" id="p-725" id="p-725" id="p-725" id="p-725" id="p-725" id="p-725" id="p-725" id="p-725"
[725] The cell-target moleingcule or pharmaceutic composial tion of the present invention may־ be administered alone or in combination with one or more other therape uticor diagnos agents.tic A combinati therapyon may include a cell- targeti moleng cule of the present invention, or pharmaceut composiical tion thereof, combined with at leas onet other therapeuti agentc selected based on the particular patient, diseas ore condi tion to be treated. Example ofs othe suchr agent includes , inter alia, a cytotoxic, anti-cance or chemotr herapeut agent,ic an anti-inflammat ory' or anti-prolifer agent,ative an antimicrobia or antivirall agent, growth factor s,WO 2016/196344 PCT/US2016/034778 263 cytokines, an analgesi a therapeutc, icall active ysmall molecule or polypepti ade, singl chaine antibody, a classica antil body or fragment there of,or a nucleic aci d molecule which modulates one or more signaling pathways and, similar modulating therapeutic molecule whichs may complement or otherwise be beneficial in a therapeutic or prophylactic treatment regimen. 726. 726. 726. id="p-726" id="p-726" id="p-726" id="p-726" id="p-726" id="p-726" id="p-726" id="p-726" id="p-726" id="p-726"
[726] Treatment of a patient with cell-targeti molengcule or pharmaceuti cal composition of the present invention preferab lealyds to cell death of targe tedcel ls and/or the inhibition of growth of targe tedcell s.As such, cytotoxic, cell-targetin g molecules of the prese ntinvention, and pharmaceuti compocal sitions comprising them., will be useful in methods for treat inga vari ety of pathological disorder in s which killing or depleting targe cellst may be beneficial such, as, inter alia, cancer , tumors othe, growthr abnormalit immuneies, disorders and, infecte celd ls. The present invention provides methods for suppressing cell proliferat andion, treati ng cell disorde includrs, ing neoplas overacia, tive B-cells, and overactive T-cells. 727. 727. 727. id="p-727" id="p-727" id="p-727" id="p-727" id="p-727" id="p-727" id="p-727" id="p-727" id="p-727" id="p-727"
[727] In certain embodiments, the cell-target moleculeing ands pharmaceutic al compositions of the present invention can be used to treat or prevent cancers, tumors (malignant and non-malignant) growth, abnormalit immuies, ne disorders and, microbial infections. In a furthe aspr ect, the above ex vivo method can be combined with the above in vivo method to provide methods of treating or preventing rejection in bone marrow' transpla recintpients and, for achieving immunological tolerance. 728. 728. 728. id="p-728" id="p-728" id="p-728" id="p-728" id="p-728" id="p-728" id="p-728" id="p-728" id="p-728" id="p-728"
[728] In certa embodimein nts, the prese ntinvention provides methods for treat ing malignancies or neoplas andms othe bloodr cel assl ociate cancersd in a mammalian subjec t,such as a human the, method comprisin theg step of administeri tong a subject m need thereof a therapeutically effective amount of a cytotoxic cell- targeting molecule or pharmaceutic compoal siti ofon the prese ntinvention. 729. 729. 729. id="p-729" id="p-729" id="p-729" id="p-729" id="p-729" id="p-729" id="p-729" id="p-729" id="p-729" id="p-729"
[729] The cell-targetin molecg ules and phannaceuti compocal sitions of the present invention have varied applications, including, e.g., uses in removing unwante T-d cell uses, s in modulating immune responses to treat graft vers ushost uses, as antiviral agents, uses as antimicrobia agelnts, and uses in purging transplanta tion tissues of unwante celd typesl The. cell-targetin molecg ules and phannaceutica l compositions of tire present invention are commonly anti-neoplastic agents --- meaning they are capable of treat ingand/o prever nting the developme maturnt, ation, or spread of neoplas ortic maligna cellsnt bv inhibiting the growth and/or causing the death of cancer or tumor cells.WO 2016/196344 PCT/US2016/034778 264 730. 730. 730. id="p-730" id="p-730" id="p-730" id="p-730" id="p-730" id="p-730" id="p-730" id="p-730" id="p-730" id="p-730"
[730] In certain embodiments, die cell-target moleingcule or pharmaceut ical composition of the present invention is used to treat a B-cell- plas, ma cell or- antibody- mediated disease or disorder, such as for example leukemia, lymphom a, myeloma, Human Immunodeficienc Virusy -rela disetedase amylois, dosis hemolyti, c uremic syndrom polyarte, eri septictis, shock, Crohn’s Disease rhe, umat oidarthrit is, ankylos spondylitis,ing psoria ticarthritis ulcerative, colitis, psoriasis ast, hma, Sjorgr’ens syndrom grafe, t-versus disease,-host graft rejection, diabetes vasc, uliti s, scleroderma and syste, mic lupus erythematosus. 731. 731. 731. id="p-731" id="p-731" id="p-731" id="p-731" id="p-731" id="p-731" id="p-731" id="p-731" id="p-731" id="p-731"
[731] In another aspect, certa embodimein nts of the cell-targetin moleculeg ands pharmaceuti compocal sitions of the prese ntinvention are antimicrobia agentsl ״ meaning they are capable of treat ingand/o prever nting the acquisiti on,development, or consequences of microbiological pathogenic infections, such as caused by viruses, bacter fungi,ia, prions, or protozoans. 732. 732. 732. id="p-732" id="p-732" id="p-732" id="p-732" id="p-732" id="p-732" id="p-732" id="p-732" id="p-732" id="p-732"
[732] It is within the scope of the prese ntinvention to provide a prophylaxis or treatment for disease ors conditions mediated by T-cells or B-cells by administerin g tiie cell-targeti molecng ule the present invention, or a pharmaceuti composcal ition thereof, to a patient for the purpose of killing T-cells or B-cells in the pati ent. This usag eis compatible wit hpreparing or conditioni a ngpatient for bone marrow transplanta stemtion, cell transplanta tistion,sue transplanta ortion, organ transplanta regartion,dless of tire sourc ofe the transplanted material, e.g. human or non-hum ansources. 733. 733. 733. id="p-733" id="p-733" id="p-733" id="p-733" id="p-733" id="p-733" id="p-733" id="p-733" id="p-733" id="p-733"
[733] It is within the scope of the prese ntinvention to provide a bone marrow recipient for prophylaxis or treatmen of host-t versus-g diseasraft viae the targe ted cell-killing of host T-cells using a cytotoxic cell-targeti molengcule or pharmaceuti composical tion of the present invention. 734. 734. 734. id="p-734" id="p-734" id="p-734" id="p-734" id="p-734" id="p-734" id="p-734" id="p-734" id="p-734" id="p-734"
[734] Certa embodin imen ofts the cell-target moleculesing and pharmaceutic al compositions of the prese ntinvention can be utilize ind a method of treating canc er comprising administerin to ag patient, in need thereof, a therapeutically effecti ve amount of a cell-targetin molegcule and/o pharmr aceutica composl ition of the present invention. In certain embodiments of the methods of the present invention, tire cance beingr treate is dselected from the group consist ingof: bone cance (sucr h as multiple myeloma or Ewin’gs sarcoma) breas, canct er, central/periphera nervousl system cancer (such as brain cancer, neurofibroma tosisor gliobla, stoma ), gastrointest canceinal (sucr h as stomach cance orr colore ctalcance r),germ cell WO 2016/196344 PCT/US2016/034778 265 cance (sucr h as ovarian cancer ands testicul canarcer glandulars, cance (sur ch as pancrea canctic er, parathyroid cancer, pheochromocytoma salivary, gland cancer, or thyroid cancer), head-neck cance (sucr h as nasopharynge cancaler, oral cancer, or pharyngea cancl er) hematol, ogical cancers (such as leukemia, lymphoma, or myeloma) kidney-u, rinary trac cancet (sucr h as renal cance andr bladder cancer), liver cancer, lung/pleura cance (sur ch as mesothelioma, smal celll lung carcinoma , or non-small cel lungl carcinoma) prosta, cancte er, sarcoma, (such as angiosarcoma, fibrosarcoma Kapos, ’si sarcoma, or synovi sarcoma)al skin, cance (sur ch as basa l cell carcinom squamousa, cell carcinoma, or melanoma) and, uterine cancer. 735. 735. 735. id="p-735" id="p-735" id="p-735" id="p-735" id="p-735" id="p-735" id="p-735" id="p-735" id="p-735" id="p-735"
[735] Certa embodin imen ofts the cell-target moleculesing and pharmaceutic al compositions of the prese ntinvention can be utilize ind a method of treating an immune disorder comprisin admig nisterin to ag patient, in need there of,a therapeutica effllyect iveamount of the cell-target molecing ules and/or pharmaceutical composition of the present invention. In certain embodiments of the methods of the present invention, the immune disorder is related to an inflamma tion associated with a diseas selece ted from the group consist ingof: amyloidosis, ankylos spondylitis,ing asthma, Crohn’s disease, diabetes graf, reject tion, graft-vs .- host disease, Hashimoto’s thyroidit hemolytiis, urec mic syndrome, HIV-related diseases, lupus erythematosu multiples, scleros polyartis, eri psoriatis, sis psoria, tic arthritis rheuma, toid arthritis scleroderma, septi, shock,c Sjorgren’s syndrom e, ulcerative colitis, and vasculitis. 736. 736. 736. id="p-736" id="p-736" id="p-736" id="p-736" id="p-736" id="p-736" id="p-736" id="p-736" id="p-736" id="p-736"
[736] Among certain embodiments of the prese ntinvention is using the Shiga toxin effector polypeptide or cell-target moleculeing of the prese ntinvention as a componen oft a pharmaceut composiical tion or medicament for the treatment or prevention of a cancer, tumor, other growth abnormality, immune disorder, and/or microbial infection. For example, immune disorders presenting on the skin of a patient may be treated with such a medicame innt efforts to reduce inflammation. In another example, skin tumors may be treated with such a medicament in effor tots reduce tumor size or eliminate the tumor completely. 737. 737. 737. id="p-737" id="p-737" id="p-737" id="p-737" id="p-737" id="p-737" id="p-737" id="p-737" id="p-737" id="p-737"
[737] Certain cytotoxic cell-target moleculeing ofs the present invention, and compositions thereof, may be used in molecul neurosurgerar apply ications such as immunolesioning and neuronal tracing (see, Wiley R, Lapp! D, Adv Drug Deliv Rev 55: 1043-54 (2003), for review'). For example, the targeting domain may be selecte d or derived from various ligands such, as neurotransmitters and neuropeptides which, WO 2016/196344 PCT/US2016/034778 266 targe specift icneuronal cell types by bindin neurg onal surface receptor suchs, as a neuronal circui specift icG-protein coupled receptor Simil. arly, the targeting domain may be selected from or derived from antibodies that bind neurona surfl ace recepto rs.Because certa Shigain toxin effector polypepti desrobustly direc theirt own retrogr axonade altransport, certai celln -targetin moleculesg of the prese nt invention may be used to kill a neuron(s) whic hexpresses the extracellula targe rat t a sit eof cytotoxic protein injection distant from the cell body (see Llewellyn-Smith I et al., J Neurosci Methods 103: 83-90 (2000)). These targete cytod toxic molecule s of tire invention that specifica targlly neuronalet cell types have uses in neuroscienc e resear ch,such as for elucidating mechanisms of sensations (see e.g. Mishra S, Hoon M, Science 340: 968-71 (2013), and creating model systems of neurodegenera tive diseases, such as Parkinson’s and Alzheimer’s (see e.g. Hamlin A et ah, PLoS One 653472 (2013)). 738. 738. 738. id="p-738" id="p-738" id="p-738" id="p-738" id="p-738" id="p-738" id="p-738" id="p-738" id="p-738" id="p-738"
[738] Among certa embodin iment of the prese ntinvention is a method of using a Shiga toxin effector polypepti cell-de, target molecing ule, pharmaceuti cal composition, and/o diagnostr compositionic of the present invention to labe orl detec thet interiors of neoplas cellstic and/o immr une cell type s.This method may be base don the ability of certai cell-n target moleculesing of the prese ntinvention to ente specifr iccei ltypes and route withi cellsn via retrograde intracell tranularspor t, to the inter iorcompartm entsof specif iccell types are labeled for detection This. can be performed on cells in situ within a patient or on cells and tissues removed from an organism e.g., biopsy material. 739. 739. 739. id="p-739" id="p-739" id="p-739" id="p-739" id="p-739" id="p-739" id="p-739" id="p-739" id="p-739" id="p-739"
[739] Among certa embodin iment of the prese ntinvention is a method of using a Shiga toxin effector polypeptide, cell-target molecing ule, pharmaceut ical composition, and/o diagnostr compositionic of the prese ntinvention to dete ctthe presence of a cell type for the purpose of information gather regardiing diseng ases , conditions and/or disorde rs,lire method comprises contacting a cell with a diagnostic sufficieally amountnt of a cell-targeti molecng ule of the prese ntinvent oni in order to detec thet molecule by an assay or diagnosti techc nique The. phra se ‘־diagnostic sufficieally amountnt " refe rsto an amount that provides adequate detectio andn accura meate surement for informati gatherion purposesng by the particula assray or diagnostic technique utilize d.Generally, the diagnostically sufficient amount for whole organism in vivo diagnos usetic will be a non- cumulative dose ofbetween 0.001 to 10 milligra ofms the detect ionpromoting agen tWO 2016/196344 PCT/US2016/034778 267 linked cell-targeti molengcule of the invention per kg of subject per subject.
Typically, the amount of Shiga toxin effector polypeptide or cell-target moleculeing of the invention used in these informati gatheron mething ods will be as low as possible provide thatd it is stil al diagnostic sufallyficient amoun t.For example, for in vivo detection in an organism, the amount of Shiga toxin effector polypeptide , cell-targetin molecg ule, or pharmaceut composiical tion of the invention administere to ad subject will be as low as feasibly possible. 740. 740. 740. id="p-740" id="p-740" id="p-740" id="p-740" id="p-740" id="p-740" id="p-740" id="p-740" id="p-740" id="p-740"
[740] The cell-type specif ictargeting of cell-target moleculesing of the present invention combine witd hdetection promoting agents provides a way to detec andt image cells physically coupled wit han extracellula target rbiomolecul of ea binding region of the molecule of the invention. Imaging of cells using the cell-targetin g molecules of the prese ntinvention may be performed in vitro or in vivo by any suitable technique known in tire art. Diagnos ticinformati mayon be collect usinged vari ous methods knowm in the art, including whole body imaging of an organism, or using ex vivo samples taken from an organism. The term "sample use" d herein refers to any number of things but, not limite to,d fluids such as blood, urine, serum, lymph, saliva, anal secretions, vaginal secretions, and semen, and tissues obtained by biopsy procedures. For example, various detection promoti agentsng may be utilized for non-invasive in vivo tumor imaging by techniques such as magnetic resonance imaging (MIU), optica methl ods (such as direct, fluoresce andnt, bioluminescent imaging), positron emission tomograph (PET),y single-photon emission computed tomography (SPECT), ultrasoun x-rayd, comput edtomography, and combinations of the aforementi oned(see, Kaur S et al., Cancer Lett 315: 97-111 (2012),/or review). 741. 741. 741. id="p-741" id="p-741" id="p-741" id="p-741" id="p-741" id="p-741" id="p-741" id="p-741" id="p-741" id="p-741"
[741] Among certa embodin iment of the prese ntinvention is a method of using a Shiga toxin effector polypepti cell-de, target molecing ule, or pharmaceuti cal composition of the present invention in a diagnosti composic tion to labe orl dete ct the interiors of a hematologi celc l,cance celr l,tumor cel l,infecte celd l,and/or immune cell (see e.g., Koyama Y et al., Clin Cancer Res 13: 2936-45 (2007); Ogawa M et al., Cancer Res 69: 1268-72 (2009); Yang L et al., Small 5: 235-43 (2009)). Based on the ability of certai cell-n target molecing ules of the invention to ente specifr iccel typesl and route within cells via. retrograde intracellula transr port, the interior compartme ofnts specif icceil types are labeled for detection. This can WO 2016/196344 PCT/US2016/034778 268 be performed on ceils in situ within a patient or on cells and tissues removed from a!1 organism e.g., biopsy material. 742. 742. 742. id="p-742" id="p-742" id="p-742" id="p-742" id="p-742" id="p-742" id="p-742" id="p-742" id="p-742" id="p-742"
[742] Diagnos ticcompositions of the prese ntinvention may be used to characteriz e a disease, disorder, or condition as potentially treatable by a related pharmaceutic al composition of the present invention. In certa compoin sitions of matte ofr the present invention may be used to determi whetherne a patient belongs to a group that responds to a therapeuti strcateg whichy makes use of a compound, composition or related method of the prese ntinvention as described herein or is well suite dfor using a delivery devic ofe the invention. 743. 743. 743. id="p-743" id="p-743" id="p-743" id="p-743" id="p-743" id="p-743" id="p-743" id="p-743" id="p-743" id="p-743"
[743] Diagnosti c compositions of the prese nti nvention m ay be used after a disease, e.g. a cancer, is detected in order to better characte it,rize such as to monitor distant metastas heterogeneies, andty, stage of cancer progress ion.Hie phenotypi c assessment of diseas disordee orr infection can help prognostic and prediction during therapeut decisionic making. In diseas reoce currence certa, methodsin of the invention may be used to determine if loca orl systemic problem. 744. 744. 744. id="p-744" id="p-744" id="p-744" id="p-744" id="p-744" id="p-744" id="p-744" id="p-744" id="p-744" id="p-744"
[744] Diagnostic compositions of the prese ntinvention may be used to assess responses to therapies regardless of the type of the type of therapy, e.g. small molecule drug, biologica drug,l or cell-based therapy. For example, certa in embodiments of the diagnost ofics the invention may be used to measure changes in tumor size ,changes in antigen positive cell populations including number and distribution, or monitoring a differ entmarker than the antigen targete by da therap y already being administer to eda. patient (see Smith-Jones P et al., Nat. Biotechnol 22: 701-6 (2004); Evans M et al., Proc. Natl. Acad. Sci. USA 108: 9578-82 (2011)). 745. 745. 745. id="p-745" id="p-745" id="p-745" id="p-745" id="p-745" id="p-745" id="p-745" id="p-745" id="p-745" id="p-745"
[745] In certa embodimein nts of the method used to detec thet presence of a cell type may be used to gather informati regardingon diseases disor, ders and, conditions, such as, for example bone cance (sucr h as multiple myeloma or Ewing’s sarcoma) brea, stcancer, central/periphera nervousl system cancer (such as brain cancer, neurofibroma tosisor glioblas, toma) gastroi, ntest canceinal (sucr h as stoma ch cance orr colorect cancal er) germ, cell cance (suchr as ovaria cancern ands testicular cancers, glandula cancer (sucr h as pancrea canctic er, parathyroid cancer, pheochromocytoma, salivary gland cance orr, thyroid cancer) head-, neck canc er (such as nasopharynge cancaler, oral cancer, or pharyngea cancel r),hematologi cal cancer (susch as leukem ia,lymphoma, or myeloma) kidney-urina, tracry canct er (such as renal cancer and bladder cance r),liver cancer, lung/pleura cance (sucr h as WO 2016/196344 PCT/US2016/034778 269 mesothelioma smal, celll lung carcinoma, or non-sma cellll lung carcinoma) , prostate cance sar, rcoma (such as angiosarc oma,fibrosarcoma Kapos, ’si sarcoma, or synovia sarcl oma skin), cance (sucr h as basa celll carcinoma, squamous cell carcinoma, or melanoma) uter, ine cance AIDS,r, amyloidosis ankylosing, spondyli asttis, hma, autism car, diogenes Crohnis, ’s disease, diabetes erythema, tosus, gastritis, graft rejection, graft-versus-ho diseasst Gravee, ’s disease, Hashimoto’s thyroiditis, hemolytic uremic syndrome, HIV-related di seases, lupus erythematosus , lymphoprolife disorrativeders (including post-transpla lymphoprnt olife rative disorders), multiple scleros myastis, henia grav is,neuroinflamma polyarttion, eri tis, psoriasis psor, iati arthritis,c rheumatoid arthritis scle, roderm septia, shocc k, Sjorgr’ens syndrom syse, temic lupus erythematosus ulcer, ative coliti vascs, uliti s, cell proliferat inflaion, mmati leukocon, yte activation, leukocyte adhesion, leukocyte chemota xis,leukocyte maturation, leukocyte migration, neuronal differentiation, acute lymphoblast leukemiic (ALL),a T acute lymphocyti leukemiac /lymphoma (ALL), acute myelogenous leukem ia,acute myeloid leukem ia.(AML), B-cell chronic lymphocyt leukemiic (B-a CLL), B-cell prolymphocytic lymphoma, Burkitt’s lymphom (BL),a chronic lymphocyti leukec mia (CLL), chronic myelogenous leukemi (CML-a BP), chronic myeloid leukemi (CML)a , diffuse larg e B-cell lymphoma, follicula lymphoma,r hair cey ll leukemi (HCL),a Hodgkin’s Lymphoma (HL), intravascular large B-cell lymphoma, lymphomat oid granulomatosis lymphoplas, macytic lymphoma, MALT lymphoma, mantle cell lymphoma, multiple myeloma (MM), natura killerl cell leukemia, nodal marginal B- cell lymphoma, Non-Hodgkin’s lymphom (NHLa ), plasma cell leukemia, plasmacytoma, primar effy usion lymphoma, pro-lymphocyti leukemic a, promyeloc leukeytic mia, small lymphocyti lymphoma,c splenic marginal zone lymphoma, T-cell lymphom (TCL),a heavy chain disease, monoclonal gammopat hy, monoclonal immunoglobulin deposition disease, myelodusplas syndrometic s (MDS), smoldering multiple myeloma, and Waldenstrom macroglobuline mia. 746. 746. 746. id="p-746" id="p-746" id="p-746" id="p-746" id="p-746" id="p-746" id="p-746" id="p-746" id="p-746" id="p-746"
[746] In certa embodimein nts, the Shiga toxin effecto polypeptidesr and cell- targeti moleculesng of the prese ntinvention, or pharmaceut compositionsical there of,are used for both diagnosi ands treatment, or for diagnos alone.is In some situations, it would be desirable to determine or verify the HLA variant(s) and/or HLA alleles express ined the subjec and/ort diseased tissue from the subject such, as, WO 2016/196344 PCT/US2016/034778 270 e.g.. a patient in need of treatment, befor selecte inga Shiga toxin effect or polypeptide or cell-targeti molecng ule of the invention for use in treatment(s ). 747. 747. 747. id="p-747" id="p-747" id="p-747" id="p-747" id="p-747" id="p-747" id="p-747" id="p-747" id="p-747" id="p-747"
[747] Any embodime ofnt the Shiga, toxin effector polypeptide of the present invention and cel I-targeting molecule of the prese ntinvention (e.g. embodimen ofts embodiment sets #1-11 in the Summar7)y may be used with eac hindividual embodime ofnt the methods of the present invention. 748. 748. 748. id="p-748" id="p-748" id="p-748" id="p-748" id="p-748" id="p-748" id="p-748" id="p-748" id="p-748" id="p-748"
[748] 'The present invention is furthe illur strate by thed followi non-limng iting examples of 1) Shiga toxin effector polypeptides of the present invention, 2) cell - targeting molecules of the prese ntinvention, and 3) cytotoxic, cell-targeting molecules of the prese ntinvention comprisin theg aforementioned polypepti desand capable of specifica targetlly certaining cell types.
EXAMPLES 749. 749. 749. id="p-749" id="p-749" id="p-749" id="p-749" id="p-749" id="p-749" id="p-749" id="p-749" id="p-749" id="p-749"
[749] The following examples demonstrat ceretai embodimn ents of the prese nt invention. Howeve itr, is to be understood that these examples are for illustrati on purposes only and do not intend, nor should any be construed, to be wholly definit iveas to conditions and scope of this invention. The experiment in thes following examples were carri edout using standa techniqurd whiches, are well known and routine to those of skill in the art, except where otherw described.ise 750. 750. 750. id="p-750" id="p-750" id="p-750" id="p-750" id="p-750" id="p-750" id="p-750" id="p-750" id="p-750" id="p-750"
[750] The followi examng ples describe several, exempla ry,cytotoxic, Shiga toxin A Subunit derived polypeptide scaffolds comprisin Shigag toxin effector polypepti desof the prese ntinvention. The Shiga toxin effector polypepti desin the Examples are de-immuniz edwhile retaining catalyti and/oc cytor toxic activities. 751. 751. 751. id="p-751" id="p-751" id="p-751" id="p-751" id="p-751" id="p-751" id="p-751" id="p-751" id="p-751" id="p-751"
[751] The following examples als odescribe several, cytotoxic, cell-targetin g molecule eacs, hmolecule comprisi nga Shiga toxin effecto polypeptider linked, either directly or indirectly, to a cell-targetin binding regg ion capabl ofe bindin ang extracellular part of a targe biomt olec ulephysical associly ated wit ha cellular surfa ceof a cell Exemplar. 7, cytotoxic,y cell-targeti molecng ules describe belowd bound to cell-surf ace,targe biomolt ecule expres ss byed targeted, tumor cell-ty pes and entered those targete ceild s.Tire internal izecelld, -targetin molecg ules effective routely theid Shigar toxin effector polypepti desto the cytosols of targe t cells where the Shiga toxin effector polypepti desinactivate ribosd omes and subsequently cause thed apoptotic death of the target celled s.The exemplar celly­WO 2016/196344 PCT/US2016/034778 271 targeti moleculesng of the invention are able to effective delivly erimmunogenic, T- cell epitop esto the MHC clas Is pathway of targe cells.t 752. 752. 752. id="p-752" id="p-752" id="p-752" id="p-752" id="p-752" id="p-752" id="p-752" id="p-752" id="p-752" id="p-752"
[752] Additiona lly,some of the exemplar cell-y' target molecing ules comprise protease-cle avageresistant, de-immunize Shigad, toxin effector polypepti desthat exhibite improved ind vivo immunogenicit profiy les (reductions in antibody responses) as compared to parenta cy totoxicl molecules comprisin a gfurin-cleava ge resistan Shigat, toxin effector polypeptide that had not been further de-immuniz ed by the disruption of additiona endogel, nous epitope regions Furthermore,. thes e exempla ry,protease-cle avageresistant, de-immuniz edcell-targetin moleculeg s exhibit improv edin vivo tolerability as compared to related cell-targetin moleculeg s comprising more protease-cleava sensigetive Shiga toxin effecto polypeptr ide regions. 753. 753. 753. id="p-753" id="p-753" id="p-753" id="p-753" id="p-753" id="p-753" id="p-753" id="p-753" id="p-753" id="p-753"
[753] The Examples below describe certain, Shiga toxin effector polypepti desof the prese ntinvention and their properti es.Certa Examplesin describe de- immunized, Shiga, toxin effecto polyper ptide of thes present invention that compri se embedded, heterologous CD8+, T-cell epitopes Certa. Examplein describes de- immunized, Shiga toxin effector polypeptides of the present invention that are furin- cleava resistge ant. Certain Examples describe furin-cleava resistge ant, de- immunized, Shiga toxin effecto polyper ptide of thes present invention that compri se embedded, heterologous CD8+, T-cell epitopes Certa. Examplesin describe furin- cleava resge ista Shigant, toxin effecto polypeptidesr of the prese ntinvention that comprise embedded, heterologous, CD8+ T-cell epitopes wit honly minimal de- immunization. Furthermor thee, Examples belo wdescribe certai celln, -targetin g molecules of the prese ntinvention and thei properties.r Certa Examplesin describe cell-target moleculeing ofs the present invention wherein a Shiga toxi neffector polypeptide componen (1)t is de-immunize (2)d; is on or proximal to an amino- termin ofus a polypeptide component of the cell-targeti moleculeng (3); is furin- cleava resge ista and/ont; (4)r compris anes embedded or inserted T-cell epitope.
Certa Examplesin descri becell-targeting molecules wherein a polypeptide component of the cell-targeting molecule compris aes carboxy-termi nal, endoplasmic reticulum retention/re trievasignal motif.lWO 2016/196344 PCT/US2016/034778 272 Example 1. Identifying Endogenous, Epitope Regions in Shiga Toxin A Subunit Effector Polypeptides 754. 754. 754. id="p-754" id="p-754" id="p-754" id="p-754" id="p-754" id="p-754" id="p-754" id="p-754" id="p-754" id="p-754"
[754] Polypeptide sequences of the A Subunits from multiple Shiga toxins of the Shiga toxin family were analyz toed identify putative, antige nicand/or immunogeni c epitopes This. Example shows how antigenic and immunogeni epitc opes can be identif iedin Shiga toxin A Subunits and related polypepti des(see also WO 2015/113005: WO 2015/113007). Computational methods were used to predict antigenic and/o immunor geni epitc opes in various Shiga toxin A Subunits includ, ing utilizing publicly availa bledata regarding protein structures. Both B-cell epitopes and CD4+ T-cell epitopes wit ha potentia to elil cit immune response weres predicte d in silieo. Epitope predictions wer evalidated empirica lly(see Example 2, infra; WO 2015/113 005; WO 2015/113 007). 755. 755. 755. id="p-755" id="p-755" id="p-755" id="p-755" id="p-755" id="p-755" id="p-755" id="p-755" id="p-755" id="p-755"
[755] Linear B-ce, ll epitop eswer predice ted for the mature A. Subunit of Shiga-like toxin 1 (SLT-1 A; SEQ ID NO:1) from the polypeptide sequence and 3D structura l data of Shiga-Like Toxin Chain A (PDB ID: 1DM0 A) by Prolmmune Inc.
(Sarasota, FL, U.S.) using thei REVEAr L® system. 756. 756. 756. id="p-756" id="p-756" id="p-756" id="p-756" id="p-756" id="p-756" id="p-756" id="p-756" id="p-756" id="p-756"
[756] In addition, B-cell epitop eswer epredicted in the polypeptide sequence ofs the A Subunit sof Shiga toxin (StxA; SEQ ID NO:2), Shiga-like toxin I (SLT-1A; SEQ ID NO: 1), and Shiga-like toxin 2 (Stx2A ;SEQ ID NO:3) using the BcePred webserver (Saha S, Raghava G, Lecture Notes in Comput Set 3239: 197-204 (2004)), Bepipred Linear Epitope Prediction (Larsen J et al., Immunome Res 2: 2 (2006)), and ElliPro Antibody epitope predicti (Haston Andersee Pn et al., Protein Sci 15: 2558-67 (2006); Ponomarenko J, Bourne P, BMC Struct Biol 1; 64 (2007)).
The various computational methods reveal similaed predicr tions for B-cell epitope regio nsin three prototypi, Shigacal, toxi An Subunit s(Table s1-3).WO 2016/196344 PCT/US2016/034778 273 Table 1. B-Cel lEpitope Predictio forns the Mature, Native A Subunit of Shiga- like Toxin 1 (SEQ ID NO:1) native positionedly amino aci dpositions REVEAL BcePredBepipred ElliPro 29-35 28-34 27-37 42 48 39 46 43 47 58-66 56-64 57-66 55—61 96-103 105-111 100-115 96-110 144-151 141-147 147-151 144-153 183-189 181-187 183-185 180-190 211-219 2.43-251 243-257 257-268 261-267 254-268 289-293 285-291 262-293 Table 2. B-Cel lEpitope Predictions for the Mature Nat, ive A Subunit of Shiga Toxin (SEQ ID NO:2) natively positioned amino aci dpositions REVEAL BcePredBepipred ElliPro 29-35 28-34 27-37 42-48 39-46 44-47 58-66 55-61 56-64 57-66 96-103 105-111 100-115 96-110 144-151 141-147 147-151 144-153 183-189 181-187 183-185 180-190 211-219 243-251 243-257 257-268 261-267 254-268 289-293 285-291 262-293 Table 3. B-Cel lEpitope Predictio forns the Mature, Native A Subunit of Shiga- like Toxin 2 (SEQ ID NO:3) natively positioned amino add positions BcePred Bepipred ElliPro 3-11 8-14 29-35 28-36 26-37 42-48 57-62 56-66 108-115 109-115 96-110 141-156 140-153 179-188 180-191 210-218 210-217 240-257 244-258 241-255 262-278 281-297WO 2016/196344 PCT/US2016/034778 274 757. 757. 757. id="p-757" id="p-757" id="p-757" id="p-757" id="p-757" id="p-757" id="p-757" id="p-757" id="p-757" id="p-757"
[757] There wer nine,e predicte B-celld, epitope regio nsin SLT-1A which were identifie byd more than one method as overlapp regionsing (Table 4).
Table 4. Putative, B-Cel lEpitope Regions in Prototypical, Shiga Toxin A Subunits natively positioned amino acid positions SLT-1A StxA SLT-2A 3-14 27-37 27-37 26-37 42 49 39-48 39-48 | 56-66 55-66 55-66 96-115 96-115 96-115 141-153 141-153 140-156 180-190 180-190 179-191 210-218 243-257 243-257 240-260 254-268 254-268 262-278 285-293 285-293 281-297 758. 758. 758. id="p-758" id="p-758" id="p-758" id="p-758" id="p-758" id="p-758" id="p-758" id="p-758" id="p-758" id="p-758"
[758] In addition, Shiga toxin A Subunits wer eanalyzed using the Epitopia webserver for predicting B-cell epitopes and immunogeni resc idues (Rubinstein N et al., BMC Bioinformatics 10: 287 (2009)). Epitopia was used to identify linea r, amino acid residue regions predicted to be immunogeni inc SLT-1A base don an Epitopia score of 4 or 5 ("high") for the major ityof amino acid residues within a linear, amino acid residue region. 759. 759. 759. id="p-759" id="p-759" id="p-759" id="p-759" id="p-759" id="p-759" id="p-759" id="p-759" id="p-759" id="p-759"
[759] The Epitopia analysis predicted an immunogeni regc ion occurs from amino acid residues 1 to 15 in SET-LA (designate as d"Epitope Region 1", see Table 5).
Based on the Epitopia analysis the, immunogeni epitopec region 39-48 in SLT-1A (see Table 4) might include position 49 (designa tedas "Epitope Region 3", see Table 5). Base don the Epitopia analysis the, immunogeni epitopec region 55-66 in SLT-1A (see Table 4) might include position 53 and extend to around position 62- 66 (designa tedas "Epitope Region 4", see Table 5), the epitope region 96—115 in SLT-1A (see Table 4) might include position 94 (designa tedas "Epitope Region 5", see Table 5), and the epitope region 180-190 in SLT-1A (see Table 4) might sta rtat position 179 and extend to around position 188-190 (designate as d"Epitope Region 7", see Table 5).WO 2016/196344 PCT/US2016/034778 275 Table 5. Prototypical Shiga, Toxin A Subunits Share Ten, Putative B-Cel, l 760. 760. 760. id="p-760" id="p-760" id="p-760" id="p-760" id="p-760" id="p-760" id="p-760" id="p-760" id="p-760" id="p-760"
[760] T-cell epitopes were predicted for the mature, A Subuni tof Shiga-like toxin 1 (SEQ ID NO:1) by the REVEAL™ Immunogenici Systemty (IS) T-cell assa y performed by Prolmmune, Inc. (Sarasota, FL, U.S.). This assay uses multiple, overlapping peptides from a protein of interes to ttes fort the elicitati ofon any immune respons bye mammalian, CD4+ T-cells from healthy donor cell samples depleted of CD8+ T-cells. Prolmmune’s REVEAL™ assa predicy ted seven, T-cell epitopes in SLT-1A (Tabl e6).
Table 6. Putative, CD4+ T-Cell Epitopes in a Prototypica Shigal, Toxin A Subunit natively positioned amino aci dpositionsT-cel epitopel # 1 4-33 34-78 2 3 77-103 4 128-168 160-183 236-258 6 ר 274-293 15WO 2016/196344 PCT/US2016/034778 276 761. 761. 761. id="p-761" id="p-761" id="p-761" id="p-761" id="p-761" id="p-761" id="p-761" id="p-761" id="p-761" id="p-761"
[761] All of the ten, predicte B-ced, ll epitope regio ns(Table 5) overlapped with at lea stone, CD4+ T-cell epitope predict byed the REVEAL™ assay (Table 7).
Table 7. B-Cel iEpitope Regions in Prototypical Shiga, Toxin A Subunits _Overl apwit hPredicte CD4+d, T-Cel lEpitopes_____ nativel posity ioned amino add positions Epitope Region B-Cel lEpitope Region T-Cel lEpitope(s) 1-15 4-33 1 2 27-37 4-33;34-78 | 3 39-48 34-78 4 53 66 34-78 | 5 94-115 77-103 6 141-153 128-168 7 179-190 160-183 243-257 236-258 8 9 254-268 236-258 285-293 274-293 762. 762. 762. id="p-762" id="p-762" id="p-762" id="p-762" id="p-762" id="p-762" id="p-762" id="p-762" id="p-762" id="p-762"
[762] In order to improve Shiga toxin-derived polypeptides for therapeutic and diagnosti applc ications in chordate differents, Shiga, toxin A Subunit effector polypepti deswere construct to edbe de-immuniz edand furin-cleavage resistant, as wel asl in some instanc toes comprise an embedded heterol, ogous, CD8+ T-cell epitope (referre to dherei asn "CD8+ T-cell hyper-immunized"). CD8+ T-cell epitopes wer eembedded or insert intoed Shiga toxin effecto polypeptidesr by engineerin interng sub-regionsal of Shiga toxin effector polypeptides to comprise a heterologous T-ce,ll epitope (see e.g. WO 2015/113007). The embedding or inserting of heterologous, T-cell epitopes may be used to disrupt an endogenous, B- cell and/or CD4+ T-cell epitope region(s in) order to further de-immunize a Shiga toxin A Subunit derived scaffold (WO 2015/113007). All of the predicte B-ced, ll epitope regions and T-cell epitopes in Table 7 wer edisrupte and/ord delete d individual orly in combinati inon the followi examplng es. 20WO 2016/196344 PCT/US2016/034778 277 Example 2. Construct anding Testing Exemplar Shigay, Toxin Effector Polypepti desand Cell-Target Molecing ules of the Present Invention 763. 763. 763. id="p-763" id="p-763" id="p-763" id="p-763" id="p-763" id="p-763" id="p-763" id="p-763" id="p-763" id="p-763"
[763] This example describes the creation and testing of various scaffolds comprising Shiga toxin effecto polypeptidesr which are de-immunize suchd, as, e.g., as shown by reductions in antigenicity and/o immr unogenicit relaty iveto other Shiga toxin effector polypeptide Ins. addition, some of the Shiga toxin effector polypepti desof this Example are more protease-clea resvageistan thant wild-type Shiga toxin effector polypepti desand/o compriser embedded or inserte d, heterologous CD8+, T-cell epitopes The. Shiga toxin effecto polypeptidesr of this Example wer testee asd compone ntsof vari ous, cell-targeti molecng ules of the present invention.
Construction of Exempla ry,Shiga Toxin Effector Polypeptide (SLT-lA-s combo(n)) and Cell-Target Molecing ules Comprising the Same (SLT-lA-combo(n)::scFv-(n)) 764. 764. 764. id="p-764" id="p-764" id="p-764" id="p-764" id="p-764" id="p-764" id="p-764" id="p-764" id="p-764" id="p-764"
[764] De-immunized, Shiga toxin A Subunit effector polypeptides wer ecreate d and teste ind the context of cell-targetin molegcule eachs, comprisin a gcell - targeting, immunoglobulin-type binding regio linkedn to a Shiga toxin effector polypeptide region. 765. 765. 765. id="p-765" id="p-765" id="p-765" id="p-765" id="p-765" id="p-765" id="p-765" id="p-765" id="p-765" id="p-765"
[765] To engineer protease-cleava resistage nceinto Shiga toxin A Subunit derived polypeptide thes, amino acid residue substitutions, R248A and/o R25r 1 A, were introduced into Shiga toxin effector polypepti des(see e.g. WO 2015/191764). 'The R248A and R251A substitutions, either individually or in combinatio disrn, upt the furin-cleava motifge at the carboxy-terminus of the Shiga toxin Al fragment regio n and represent one or more mutations in tire minima l,furin-cleava motifge relati tove a wild-type Shiga toxin A Subunit (see WO 2015/191764). 766. 766. 766. id="p-766" id="p-766" id="p-766" id="p-766" id="p-766" id="p-766" id="p-766" id="p-766" id="p-766" id="p-766"
[766] For this Example, R248A and R251A wer eintroduced into a Shiga toxin effector polypept idederived from the A subunit of Shiga-like Toxin 1 (SLT-1A) comprising amino acid s1—251 of SLT-1A (SEQ IDNO:4). The SLT-1A 1-251 R248A/R251A double mutant (SEQ ID NO:5) is referre to dherein as furin-cleava ge resist antSLT-1A or more simpl y"SLT-1A-FR." The disrupti onof the minimal, furm-cleavage site R-x-x-Rint hefurin-cleava motifge at the carboxy-termi ofnus the Shiga toxin Al fragment regio withn R248A and R251A results in decreased cleava byge furi n(see Example 3, injra; WO 2015/191764). The disruption of the minimal furm-cleavage site R-x-x-R in tire furin-cleavage motif natively positioned WO 2016/196344 PCT/US2016/034778 278 from amino acid residues 238 to 257 in StxA and SLT-1A was predicted to decreas e tire sensitivi ofty this regio ton proteol ysisby othe proter ase suchs as, e.g., proprotein convertas andes highly promiscuous protease Ins. addition, the R248A and/o R251Ar mutations disrup (1)t B-cell epitope region #8, which is native ly positioned at amino acid residues 243-259 in StxA and SLT-1A, and (2) a CD4+ T- cell epitope natively positioned at amino acid residues 236-258 in StxA and SLT- 1A. 767. 767. 767. id="p-767" id="p-767" id="p-767" id="p-767" id="p-767" id="p-767" id="p-767" id="p-767" id="p-767" id="p-767"
[767] De-immunize d,Shiga toxin effector polypepti deswere creat byed adding multipl amie, no acid residue substitutions to disrupt predicted B-cell and/o CD4+r T-cell epitope regions, including modifications result ingin embedded, heterologous, CD8+ T-cell epitopes (see Table 8; WO 2015/113005; WO 2015/113007). In order to create Shiga toxin effector polypeptides that were furthe de-ir mmunized, SLT- 1A-FR (SEQ ID NO:5) was modified to include multiple, amino acid residue substitutions to disrupt predicted B-cell and/o CD4+r T-cell epitope regions , including modifications result ingin embedded, heterologous CD8+, T-cell epitopes (see Table 8; WO 2015/113005; WO 2015/113007). Table 8 shows twent y, different, de-immunize proteasd, e-cle avageresistant, Shiga toxin A. Subunit effector polypepti desname dSLT-lA-combo(n), where n represe ntsan integ ersuch as 0, 1, 2, 3, etc. to denote differ entvariations. The numbering of the endogenous epitope regio nsmentioned in Table 8 is in accord with tire numbering schem ine Tables 6-7.
The Shiga toxin effecto polyper ptide in Tables 8 wer tese ted as described in late r sections.
Table 8. Exempla ry,De-immunized, CD8+ T-Cell Hyper-Immunized, Shiga Toxin Effector Polypeptide of thes Present Invention SLT-1A- SequenceSummary Description c0mb0(n) name epitope region 5 disrupted by amin oaci dsubstitutions epitope region 5 disrupted by embedde d,T-cell epitope epitope region 8 disrupted by amin oaci dsubstitutions SLT-1A- SEQ ID epitope regions 8, 9, and 10 disrupted by truncation comboO NO ;6 furin-cleavage motif disrupted by R248A/R251A T-cell epitope #6 disrupted by amino aci dsubstitutions T-cell epitopes #6 and #7 disrupted by truncation epitope region 4 disrupted by amin oaci dsubstitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 8 disrupted by amin oaci dsubstitutions SLT-1A- SEQ ID epitope regions 8, 9, and 10 disrupted by truncati on combo 1 NO:7 furin-cleava motifge disrupted by R248A/R251A T-cell epitope #2 disrupted by amin oaci dsubstitutions T-cell epitope #2 disrupted by embedde d,T-cell epitopeWO 2016/196344 PCT/US2016/034778 279 T-cell epitope #6 disrupted by amino aci dsubstitutions T-cell epitopes #6 and #7 disrupted by truncation epitope region 3 disrupted by amin oaci dsubstitution epitope region 4 disrupted by amin oaci dsubstitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 8 disrupted by amin oaci dsubstitutions SEQ ID SLT-1A- epitope regions 8, 9 and 10 disrupted by truncation furin-cleavage site disrupted by R248A/R251A combo?. NO:8 T-cell epitope #2 disrupted by amino aci dsubstitutions T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitope #6 disrupted by amin oaci dsubstitutions T-cell epitopes #6 and #7 disrupte byd truncation epitope region 4 disrupted by amin oacid substitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 8 disrupted by amin oacid substitutions epitope regions 8, 9 and 10 disrupted by truncation SLT-1A- SEQ ID furin-cleava sitege disrupted by R248AZR251A NO:9 combo? T-cell epitope #2 disrupted by amin oacid substitutions T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitope #6 disrupted by amin oacid substitutions T-cell epitopes #6 and #7 disrupted by truncation epitope region 4 disrupted by amin oaci dsubstitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 8 disrupted by amin oaci dsubstitutions epitope regions 8, 9 and 10 disrupted by truncation SLT-1A- SEQ ID furin-cleavage site disrupted by R248A/R251A combo4 NO: 10 T-cell epitope H2 disrupted by amino aci dsubstitutions T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitope H6 disrupted by amino aci dsubstitutions T-cell epitopes H6 and #7 disrupted by truncation epitope region 3 disrupted by amin oaci dsubstitution epitope region 4 disrupted by amin oacid substitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 5 disrupted by amin oacid substitution SLT-1A- SEQ ID epitope region 7 disrupted by amin oacid substitution combo5 NO: Il epitope regions 8, 9 and 10 disrupted by truncation T-cell epitope #2 disrupted by amin oacid substitutions T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitopes #6 and #7 disrupted by truncation epitope region 3 disrupted by amin oaci dsubstitut ion epitope region 4 disrupted by amin oaci dsubstitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 5 disrupted by amin oaci dsubstitut ion epitope region 7 disrupted by amin oaci dsubstitut ion SLT-1A- SEQ ID epitope region 8 disrupted by amin oaci dsubstitutions combo6 NO: 12 epitope regions 8, 9 and 10 disrupted by truncation T-cell epitope H2 disrupted by amino aci dsubstitutions T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitope disrupted by amino aci dsubstitut ion T-cell epitopes H6 and #7 disrupted by truncation epitope region 3 disrupted by amin oaci dsubstitution epitope region 4 disrupted by amin oacid substitutions SLT-1A- SEQ ID epitope region 4 disrupted by embedde d,T-cell epitope combo? NO: 13 epitope region 5 disrupted by amin oacid substitution epitope region 7 disrupted by amin oacid substitutionWO 2016/196344 PCT/US2016/034778 280 epitope region 8 disrupted by substitutions furin-cleava sitege disrupte byd R248AZR251A epitope regions 8. 9 and 10 disrupte byd truncation T-cell epitope #2 disrupt edby amin oaci dsubstitutions T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitope #6 disrupted by amino aci dsubstitutions T-cell epitopes #6 and #7 disrupted by truncation epitope region 3 disrupted by amin oaci dsubstitut ion epitope region 4 disrupted by amin oaci dsubstitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 5 disrupted by amin oaci dsubstitut ion epitope region 7 disrupted by amin oaci dsubstitut ion epitope region 8 disrupted by substitutions SLT-1A- SEQ ID furin-cleavage site disrupted by R248A/R251A NO :27 combo21 epitope regions 8, 9 and 10 disrupte byd truncation T-cell epitope #2 disrupted by amino aci dsubstitutions T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitope #3 disrupted by amino aci dsubstitut ion T-cell epitope #6 disrupted by amino aci dsubstitutions T-cell epitopes #6 and #7 disrupted by truncation epitope region 3 disrupted by amin oaci dsubstitut ion epitope region 4 disrupted by amin oaci dsubstitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 5 disrupted by amin oaci dsubstitution epitope region 7 disrupted by amin oaci dsubstitut ion epitope region 8 disrupted by substitutions SLT-1A- SEQ ID furin-cleavage site disrupted by R248A/R251A NO :29 combo23 epitope regions 8, 9 and 10 disrupted by truncation T-cell epitope #2 disrupted by amino aci dsubstitutions T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitope #3 disrupted by amino aci dsubstitution T-cell epitope #6 disrupted by amino aci dsubstitutions T-cell epitopes #6 and #7 disrupte byd truncation epitope region 3 disrupted by amin oaci dsubstitut ion epitope region 4 disrupted by amin oaci dsubstitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 5 disrupted by amin oaci dsubstitution epitope region 7 disrupted by amin oaci dsubstitution epitope region 8 disrupted by substitutions SLT-1A- SEQ ID furin-cleava sitege disrupte byd־ R248A/R251A combo24 NO:30 epitope regions 8, 9 and 10 disrupted by truncation T-cell epitope #2 disrupte byd־ amino acid substitutions T-cell epitope #2 disrupte byd embedde d,T-cell epitope T-cell epitope #4 disrupte byd־ amino acid substitut ion T-cell epitope #6 disrupte byd־ amino acid substitutions T-cell epitopes #6 and #7 disrupted by truncation epitope region 3 disrupted by amin oaci dsubstitution epitope region 4 disrupted by amin oaci dsubstitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 5 disrupted by amin oaci dsubstitut ion SLT-1A- SEQ ID epitope region 7 disrupted by amin oaci dsubstitut ion combo25 NO: 31 epitope region 8 disrupted by substitutions furin-cleava sitege disrupted by R248A,-R251A epitope regions 8, 9 and 10 disrupted by truncation T-cell epitope #2 disrupted by amino aci dsubstitutionsWO 2016/196344 PCT/US2016/034778 281 T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitope #4 disrupted by amino aci dsubstitut ion T-cell epitope #6 disrupted by amino aci dsubstitutions T-cell epitopes #6 and #7 disrupted by truncation epitope region 3 disrupted by amin oaci dsubstitut ion epitope region 4 disrupted by amin oaci dsubstitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 5 disrupted by amin oaci dsubstitut ion epitope region 7 disrupted by amin oaci dsubstitut ion SLT-1A- SEQ ID epitope region 8 disrupted by substitutions furin-cleavage site disrupted by R248A/R251A combo26 NO: 3 2 epitope regions 8, 9 and 10 disrupte byd truncation T-cell epitope #2 disrupted by amin oaci dsubstitutions T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitope #6 disrupted by amin oaci dsubstitutions T-cell epitopes #6 and #7 disrupted by truncation epitope region 3 disrupted by amin oacid substitution epitope region 4 disrupted by amin oacid substitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 5 disrupted by amin oacid substitution epitope region 7 disrupted by amin oaci dsubstitut ion SEQ ID SLT-1A- epitope region 8 disrupted by substitutions furin-cleava sitege disrupt edby R248AZR251A comboS NO: 14 epitope regions 8, 9 and 10 disrupt edby truncation T-cell epitope #2 disrupted by amino aci dsubstitutions T-cell epi tope #2 disrupted by embedde d,T-cell epitope T-cell epitope #6 disrupted by amino aci dsubstitutions T-cell epitopes #6 and #7 disrupted by truncation epitope region 3 disrupted by amin oaci dsubstitut ion epitope region 4 disrupted by amin oaci dsubstitutions epitope region 4 disrupted by enrbedded, T-cell epitope epitope region 5 disrupted by amin oaci dsubstitut ion epitope region 7 disrupted by amin oaci dsubstitut ion SLT-1A- SEQ ID epitope region 8 disrupted by substitutions NO: 15 furin-cleavage site disrupted by R248A/R251A combo9 epitope regions 8, 9 and 10 disrupte byd truncation T-cell epitope #2 disrupted by amino aci dsubstitutions T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitope #6 disrupted by amino aci dsubstitutions T-cell epitopes #6 and #7 disrupted bv truncation epitope region 3 disrupted by amin oaci dsubstitut ion epitope region 4 disrupted by amin oaci dsubstitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 5 disrupted by amin oaci dsubstitut ion epitope region 6 disrupted by amin oaci dsubstitut ion epitope region 8 disrupted by substitutions SLT-1A- SEQ ID furin-cleavage site disrupte byd R248A/R251A combo 10 NO: 16 epitope regions 8, 9 and 10 disrupte byd truncation T-cell epitope #2 disrupted by amino aci dsubstitutions T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitope #4 disrupted by amino aci dsubstitution T-cell epitope #6 disrupted by amino aci dsubstitutions T-cell epitopes #6 and #7 disrupt edby truncation epitope region 3 disrupted by amin oaci dsubstitution SLT-1A- SEQ ID epitope region 4 disrupted by amin oaci dsubstitutions combo 11 NO:17WO 2016/196344 PCT/US2016/034778 282 epitope region 4 disrupted by embedde d,T-cell epitope epitope region 5 disrupted by amin oaci dsubstitut ion epitope region 7 disrupted by amin oaci dsubstitut ion epitope region 8 disrupted by substitutions furin-cleava sitege disrupte byd R248A/R251A epitope regions 8, 9 and 10 disrupte byd truncation T-cell epitope 42 disrupted by amino aci dsubstitutions T-cell epi tope 42 disrupted by embedde d,T-cell epitope T-cell epitope #6 disrupted by amino aci dsubstitutions T-cell epitopes #6 and #7 disrupt edby truncation epitope region 1 disrupted by amin oaci dsubstitut ion epitope region 3 disrupted by amin oaci dsubstitut ion epitope region 4 disrupted by amin oaci dsubstitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 5 disrupted by amin oaci dsubstitut ion epitope region 6 disrupted by amin oaci dsubstitution SLT-1A- SEQ ID epitope region 8 disrupted by substitutions furin-cleavage site disrupted by R248A/R251A combo 12 NO:18 epitope regions 8, 9 and 10 disrupte byd truncation T-cell epitope 42 disrupted by־ amino acid substitutions T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitope 44 disrupted by־ amino acid substitut ion T-cell epitope #6 disrupted by־ amino acid substitutions T-cell epitopes #6 and 47 disrupted bv tnmcation epitope region 3 disrupted by amin oaci dsubstitution epitope region 4 disrupted by amin oaci dsubstitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 7 disrupted by amin oaci dsubstitutions epitope region 7 disrupted by embedde T-ced ll epitope epitope region 8 disrupted by substitutions SLT-1A- SEQ ID furin-cleavage site disrupte byd R248A/R251A combo 13 NO: 19 epitope regions 8, 9 and 10 disrupte byd tnmcation T-cell epitope 42 disrupted by amino aci dsubstitutions T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitope 44 disrupted by amino aci dsubstitutions T-cell epitope 46 disrupted by amino aci dsubstitutions T-cell epitopes 46 and #7 disrupted by truncation epitope region 3 disrupted by amin oaci dsubstitution epitope region 4 disrupted by amin oaci dsubstitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 5 disrupted by amin oacid substitution epitope region 6 disrupted by amin oacid substitution epitope region 7 disrupted by amin oacid substitution SEQ ID SLT-1A- epitope region 8 disrupted by substitutions furin-cleava sitege disrupte byd־ R248A/R251A combo 14 NO:20 epitope regions 8, 9 and 10 disrupted by truncation T-cell epitope #2 disrupted by amin oacid substitutions T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitope #4 disrupted by amin oacid substitut ion T-cell epitope #6 disrupted by amino aci dsubstitutions T-cell epitopes #6 and #7 disrupted by tnmcation epitope region 1 disrupted by amin oaci dsubstitut ion SLT-1A- SEQ ID epitope region 3 disrupted by amin oaci dsubstitut ion epitope region 4 disrupted by amin oaci dsubstitutions combo 15 NO:2I epitope region 4 disrupted by embedde d,T-cell epitopeWO 2016/196344 PCT/US2016/034778 283 epitope region 5 disrupted by amin oaci dsubstitut ion epitope region 6 disrupted by amin oaci dsubstitut ion epitope region 8 disrupted by substitutions furin-cleavage site disrupted by R248AZR251A epitope regions 8, 9 and 10 disrupte byd truncation T-cell epitope #2 disrupted by amino aci dsubstitutions T-cell epi tope #2 disrupted by embedde d,T-cell epitope T-cell epitope #4 disrupted by amino aci dsubstitut ion T-cell epitope #6 disrupted by amino aci dsubstitutions T-cell epitopes #6 and #7 disrupt edby truncation epitope region 1 disrupted by amin oaci dsubstitut ion epitope region 3 disrupted by amin oaci dsubstitut ion epitope region 4 disrupted by amin oaci dsubstitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 5 disrupted by amin oaci dsubstitut ion epitope region 6 disrupted by amin oaci dsubstitution epitope region 7 disrupted by amin oaci dsubstitution SLT-1A- SEQ ID epitope region 8 disrupted by substitutions combo 16 NO :22 furin-cleavage site disrupted by R248A/R251A epitope regions 8, 9 and 10 disrupted by truncation T-cell epitope #2 disrupted by amino acid substitutions T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitope #4 disrupted by־ amino acid substituti on T-cell epitope #6 disrupted by־ amino acid substitutions T-cell epitopes #6 and #7 disrupted bv truncation epitope region 1 disrupted by amin oaci dsubstitutions epitope region 3 disrupted by amin oaci dsubstitut ion epitope region 4 disrupted by amin oaci dsubstitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 5 disrupted by amin oaci dsubstitution epitope region 6 disrupted by amin oaci dsubstitution epitope region 7 disrupted by amin oaci dsubstitution SLT-1A- SEQ ID epitope region 8 disrupted by substitutions combo 17 NO:23 furin-cleavage site disrupte byd R248A,-R251A epitope regions 8, 9 and 10 disrupted by truncation T-cell epitope HI. disrupted by amino aci dsubstitut ion T-cell epitope H2 disrupted by amino aci dsubstitutions T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitope H4 disrupted by amino aci dsubstitut ion T-cell epitope #6 disrupted by amin oaci dsubstitutions T-cell epitopes #6 and #7 disrupted by truncation epitope region 1 disrupted by amin oacid substitutions epitope region 3 disrupted by amin oacid substitution epitope region 4 disrupted by amin oacid substitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 5 disrupted by amin oacid substitution epitope region 6 disrupted by amin oacid substitution SLT-1A- SEQ ID epitope region 7 disrupted by amin oacid substitution NO :24 epitope region 8 disrupted by substitutions combo 18 furin-cleava sitege disrupted by R248AZR251A epitope regions 8, 9 and 10 disrupted by truncation T-cell epitope #1 disrupted by amino aci dsubstitut ion T-cell epitope #2 disrupted by amino aci dsubstitutions T-cell epitope #2 disrupte byd embedde d,T-cell epitope T-cell epitope #4 disrupted by amino aci dsubstitutionWO 2016/196344 PCT/US2016/034778 284 T-cell epitope #6 disrupted by amino aci dsubstitutions T-cell epitopes #6 and #7 disrupted by truncation epitope region 1 disrupted by amin oaci dsubstitut ion epitope region 3 disrupted by amin oaci dsubstitut ion epitope region 4 disrupted by amin oaci dsubstitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 5 disrupted by amin oaci dsubstitution epitope region 6 disrupted by amin oaci dsubstitut ion epitope region 7 disrupted by substitutions epitope region 8 disrupted by substitutions SLT-1 A- SEQ ID furin-cleavage site disrupted by R248A/R251A combo 19 NO :25 epitope regions 8, 9 and 10 disrupte byd truncati on T-cell epitope #1 disrupted by amin oaci dsubstitut ion T-cell epitope #2 disrupted by amin oaci dsubstitutions T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitope #4 disrupted by amino aci dsubstitut ion T-cell epitope #5 disrupted by amino aci dsubstitut ion T-cell epitope #6 disrupted by amino aci dsubstitutions T-cell epitopes #6 and #7 disrupted by truncation epitope region 3 disrupted by amin oacid substitution epitope region 4 disrupted by amin oaci dsubstitutions epitope region 4 disrupted by embedde d,T-cell epitope epitope region 5 disrupted by amin oaci dsubstitut ion epitope region 6 disrupted by amin oaci dsubstitut ion epitope region 8 disrupted by substitutions epitope region 8 disrupted by inserted, T-cell epitope SLT-1 A- SEQ ID furin-cleava sitege disrupted by R248A/R251A NO :26 combo20 epitope regions 8, 9 and 10 disrupted by truncation T-cell epitope #2 disrupted by amino aci dsubstitutions T-cell epitope #2 disrupted by embedde d,T-cell epitope T-cell epitope #4 disrupted by amino aci dsubstitut ion T-cell epitope #6 disrupted by amino aci dsubstitutions T-cell epitope #6 disrupted by inserted, T-cel epitopel T-cell epitopes #6 and #7 disrupt edby truncation 768. 768. 768. id="p-768" id="p-768" id="p-768" id="p-768" id="p-768" id="p-768" id="p-768" id="p-768" id="p-768" id="p-768"
[768] Each of the de-immunize Shigad, toxin effector polypeptides SLT-1A- combo(n) (SEQ ID NOs: 6-21, 23-27, and 29-32) comprise a combinat ionof one or more de-immunized sub-regions wit hone or more sub-regions that compris anes embedded, heterologous CD8+, T-cel epitol pe. Most of thes polypeptidese also have a disrupted, minima furin-cll, eavage motif at the carboxy-termina of theirl Shiga toxin Al fragment sub-regions (SEQ ID NOs: 6-10, 13-21, 23-27, and 29- 32). Computational analysis in silico predicted (1) that at least two, B-cel epitopesl present in the w ild-type Shiga toxin StxA or SLT-1 A wer eeliminate ford all of the Shiga toxin effector polypeptides combo(n) referre to din Table 8, and (2) that no new, B-cell epitop eswer ecreat ined any of the Shiga toxin effector polypepti des combo(n) listed in Table 8. The properti andes functional consequences of many of the modificatio to nsindividual sub-regions in Shiga toxin effector polypepti desWO 2016/196344 PCT/US2016/034778 285 combo(n) are describe ind Exampl 3,e WO 2015/113005, WO 2015/113007, and WO 2015/191764. 769. 769. 769. id="p-769" id="p-769" id="p-769" id="p-769" id="p-769" id="p-769" id="p-769" id="p-769" id="p-769" id="p-769"
[769] Polynucleotide encodings Shiga toxin A Subunit effecto polypeptider combinat ionconstructs wer ecreate andd fuse dto a cell-targeting, immunoglobuli n- type bindin gregion encoding constructs using techniques known to the skill ed worke r.The result ingpolynucleoti encodedes celld -targetin molegcule eacs, ha polypeptide comprisin (1)g a combination, Shiga toxin effecto polypeptr ideregio n SLT-lA-combo(n) (2), a cell-targetin binding gregio "nscFv-(n)," where n represents an integ ersuch as 1, 2, 3, etc to. denote different scFvs, and (3) a linker known in the art positioned between the Shiga toxin effecto polypeptider region and the bindin region.g 770. 770. 770. id="p-770" id="p-770" id="p-770" id="p-770" id="p-770" id="p-770" id="p-770" id="p-770" id="p-770" id="p-770"
[770] Using a bacterial express ionsystem known in the art, thes polynucle eoti des wer eused to produc ate leas twenty-t seven, combination de-i, mmunize Shigad, toxin effector polypeptides (Tabl e8; SEQ ID NOs: 6—21, 23-27, and 29-32), in the context of one or more cell-targetin molecules.g After being linked to a cell - targeti bindingng region, twenty-s ofix die twenty-seven scaffolds SLT-1 A- combo(n) produced a stable full-le, ngth, catalytical actively cell-, target molecing ule.
However the, cell-target moleculeing SLT-lA-combol 8::scFv- l(SEQ ID NO:54) showed evidence of instability because SLT-lA־combol8::scFv־l degradatio was n observe byd sodium dodecyl sulfa te(SDS), polyacrylam gelide electrophor etic (SDS-PAGE) analysis of gels loade andd ran with preparati ofons SLT-1A- combol8::scFv- andl a molecular weight ladder as a reference.
A. Testing the Ribosome Inhibition Activiti ofes Exemplary, Cell-Targeting Molecul Comprisinges Shiga Toxin Effect Polypeptideor SLT-1s A-combo(n) 771. 771. 771. id="p-771" id="p-771" id="p-771" id="p-771" id="p-771" id="p-771" id="p-771" id="p-771" id="p-771" id="p-771"
[771] The enzymat actic iviti ofes various, combination de-i, mmunized, proteas e- cleava resistge ant, Shiga toxin effector polypepti deswer etest edin the context of a cell-target moleingcule using an in vitro, ribosome inhibition assay known to the skilled worker (TNT® Quick Coupled Transcription/Transla Kit, Promegation Corp., Madison, WI, LT.S.).This cell-free in vitro,, protei transln ation assay was used to determi thene ribosome inactivati capabilon iti ofes SLT-lA-combolO:sc Fv-l (SEQ ID NO:47), SLT-1 A-combol6::scFv- (SEQl ID NO:52), SLT-1A- 00mb019::scFv-l (SEQ ID NO:55), SLT-lA-comboO::scFv-2 (SEQ ID NO:57), SLT-lA-combo2::scFv-2 (SEQ ID NO:58), SLT-lA-combo3::scFv2־ (SEQ IDWO 2016/196344 PCT/US2016/034778 286 NO:59), SLT-lA-combo4::scFv-2 (SEQ ID NO:60), and SLT-lA-combol3::scFv-2 (SEQ ID NO:62). 772. 772. 772. id="p-772" id="p-772" id="p-772" id="p-772" id="p-772" id="p-772" id="p-772" id="p-772" id="p-772" id="p-772"
[772] The ribosome activity reacti wason prepared accordi tong manufacture’s r instructions. A serie ofs 10-fol dilutionsd of the cell-targeti molengcule to be test ed was prepar ined an appropriat buffere and a serie ofs identical TNT® reacti on mixtur componentse were creat fored each dilution. Each sample in the dilution serie wass com bined with eac hof the TNT® reaction mixture alongs wit h Lucifera T7se Control DNA (Promega Corp., Madison, WI, U.S.). Hie tes samplt es wer eincubated for 1.5 hours at 30 degree Celss ius (°C). After the incubation, Luciferas Asse ay Reage nt(Promega Corp., Madison, WI, U.S.) was added to all tes t samples and the amount of lucifer aseprotei transln ation was measured by luminesce nceaccordi tong manufacturer’s instructions Three. positive controls wer eused: the wild-type, SLT-1A1 fragment (SLT-1A1-WT) (SEQ ID NO:4) and two cell-target moleculeing SLT-lA-FR::s scFv-I (SEQ ID NO:34) and SLT-1A- FR::scFv-2 (SEQ ID NO:35) comprisin theg protease-cle avageresistant, Shiga toxin effector polypeptide SLT-IA-FR (SEQ ID NO:5) (see WO 2015/191764). 773. 773. 773. id="p-773" id="p-773" id="p-773" id="p-773" id="p-773" id="p-773" id="p-773" id="p-773" id="p-773" id="p-773"
[773] The leve ofl protei synthesisn inhibiti onwas determined by non-line ar regressi analon ysis of log-transformed concentra tionsof total cel I-targeting molecule versus relat iveluminescenc units.e Using statisti softwarecal (GraphPa d Prism San, Diego, CA, U.S.), the half maxim alinhibitory concentrati (1C50)on value was calculat fored eac hsample using the Prism software function of log(inhibitor) vs. response (thre paramee ters) [Y = Bottom + ((Top - Bottom) / (1 + 10A(X - Log IC50)))] under the heading dose-response-inhib Theitio IC50n. for each sample was calculated and is shown in Table 9. In this assay, measurements of the inhibition of protein synthesis represent the ribosome inactivati actonivity of the sample molecule, which is one metr icof the catalytic activity of a Shiga toxin effector polypept ideor a Shiga toxin A Subunit. As reported in the Examples a molecule, exhi biting an IC50 within 10-fold of an IC50 exhibite byd a reference molecule is considere to dexhibit ribosome inhibition activ itycomparable to that reference molecule As. reporte in dthe Examples, a molecule exhibiting an IC50 les sthan or within 10 percent of an IC50 exhibited by a reference molecule is considere to d exhibit ribosome inhibiti onactivi ty'equivale tont that reference molecule.WO 2016/196344 PCT/US2016/034778 287 Table 9. Combination, De-Immanized Proteas, e-Cleavage Resistant, Shiga Toxin Effector Polypeptide Exhibiteds Ribosome Inhibition Activi tyComparable to a Wild-Type Shiga toxin Al Fragment Ribosome Inhibition Protein Sample IC50 (pM) 2.8 SLT-1 A-combo 10::scFv־l 3.3 ؛ SLT-1 A-combo 16:: scFv-1 SLT-1 A-combo 19;: scFv-1 2.2 | SLT-1 A-FR: :scFv-1 4.0 ؛ I 4.8 | SLT -1 A-comboO:: scF v2־ SLT- lA-combo2: :scFv-2 3.1 ؛ 7.6 | SLT- lA-combo3: :scFv-2 SLT- lA-combo4: :scFv-2 6.5 ؛ SLT -1 A-combo 13:: scFv-2 3.2 SLT-1 A-FR: :scFv-2 5.9 SLT-1A1-WT 4.8 | 774. 774. 774. id="p-774" id="p-774" id="p-774" id="p-774" id="p-774" id="p-774" id="p-774" id="p-774" id="p-774" id="p-774"
[774] The ribosome inactivatio actniviti ofes ail the Shiga toxin effector polypepti descombo(n) test edwere comparable to the catalytic activity of a wild- type Shiga toxin Al fragment (SEQ ID NO:4) and/or a SLT- 1A-FR polypept ide (SEQ ID NO:5) as a componen oft a cell-target moleingcule (Tabl e9; Figure 2).
Tire ribosome inactivati actionvitie of sSLT-1 A-combo 10::scFv- l,SLT-1A- combo 16::scFv-1, SET-1 A-combo 19::scFv-1, SLT-1 A-comboO::scFv-2, SLT-1 A- combo2::scFv-2, SLT-lA-combo4::scFv-l, and SLT-1 A-combo 13 ::scFv2 were equivalent to the catalyti activityc of a wild-type Shiga toxin Al fragment (SEQ ID NO:4) and/o ar SLT-1A-FR polypept ide(SEQ ID NO:5) as a componen oft a cell - targeting molecule (Tabl e9; Figure 2). 775. 775. 775. id="p-775" id="p-775" id="p-775" id="p-775" id="p-775" id="p-775" id="p-775" id="p-775" id="p-775" id="p-775"
[775] These results demonstrat that ethe ribosome inhibiti onactivitie of scertai n, exempla ry,combination de-immunized,, protease-cle avageresistant, Shiga toxin effector polypeptides in the context of a cell-target moleculeing wer ecomparable to the catalyti actcivity of a wild-type Shiga toxin Al fragment (SEQ ID NO:4) alone or the Shiga toxin effector polypeptide SLT-1 A-FR (SEQ ID NO:5) in the context of a cell-targeti moleculeng (Table 9; Figur e2). Thus, the catalytic activitie of s certai exemplary,n, combination, de-immunized, protease-cle avageresistant, Shiga toxin effector polypepti desin the context of a cell-target moleingcule appeared comparable in this assa toy the catalytic activity of a wild-type Shiga toxin A Subunit (Tabl e9; Figure 2). 25WO 2016/196344 PCT/US2016/034778 288 B. Testing the Targete Cytotoxicityd of Exemplary, Cell-Target Molecing ules Comprising Shiga Toxin Effect Polypeptor ides SLT-lA-combo(n) 776. 776. 776. id="p-776" id="p-776" id="p-776" id="p-776" id="p-776" id="p-776" id="p-776" id="p-776" id="p-776" id="p-776"
[776] The potenc andy specific ityof cytotoxic werity testede for combination de- , immunized, Shiga toxin effector polypeptides SLT-lA-combo(n) of the presen t invention as scaffolds for building various cell-target molecules.ing The cytotoxic activitie of sexemplary, cell-targeti molecng ules comprisin a gcombination, de- immunized, protease-cleava resistage Shigant, toxin effecto polypeptr ideSLT-1A- combo(n) were determined using a targe biomolecult posite ive cell-kill assay known to the skilled worker. Ibis target positive cell-kill assa wasy used to determine the cytotoxic activitie of svarious cell-targetin molegcule eacs, hcomprisin a gcell- targeting binding regio scn Fv-(n) genetical fusely dto one of the combinatio Shigan, toxin effector polypepti desSLTA-lA-combo( n)(SEQ ID NOs: 6-32) (see Table 8 for a representa subsetive tof such molecules to form) the cell-targeti moleculeng s SEQ ID NOs: 43-81. 777. 777. 777. id="p-777" id="p-777" id="p-777" id="p-777" id="p-777" id="p-777" id="p-777" id="p-777" id="p-777" id="p-777"
[777] The cytotoxici ofties cell-targeting molecule comprs isin de-img munize d, protease-cle avageresistant, Shiga toxin effector polypepti deswer edetermined using cells expressing, at a cellular surface, significa amountsnt of the appropria te, extracellular targe biomolect ule, such as, a target of the binding region scFv-1-8.
The immunoglobulin-derived bindin gregio nsscFv-1, scFv-2, scFv-3, scFv-4, scFv- 5, scFv-6, scFv-7, scFv-8, and scFv- 9eac hbinds wit hhigh affinity to a human targe biomolecult physice ally coupled to the cellular surface ofs certain human cells.
Tire cells used in this Example wer eimmortaliz humaned, tumor cells availa blefrom tire ATCC (Manassas VA, U.S.), National Cance Instr itute of the U.S. (Frederick, MD, U.S.), and/or DSZM (Braunschwei DE).g, The cells referre to dbelow were H929, Daudi, NCT-ADR/RES(expressing HER-2 from a transfe ctedvector), HCC1419, MDA-MB-231, MOLP-8, ST486, HDLM-2, and L1236 orrnore simply cell-lines A, B, C, D, E, F, G, H, and I, respectively. Using a method known to the skilled worker, cells from cell-line C used in this Exampl were trae nsfe ctew7ithd an expression vector and made to express a significa amountnt of cell-surfa HER-2.ce 30WO 2016/196344 PCT/US2016/034778 289 778. 778. 778. id="p-778" id="p-778" id="p-778" id="p-778" id="p-778" id="p-778" id="p-778" id="p-778" id="p-778" id="p-778"
[778] Ilie cell-kill assa wasy performed as follows Cert. ain, huma ntumor, cell- line cells wer platee (atd abou t2 to 8 x 103 cel lsper wel l)in 20 microliter (pL) cel l cultur mediume in 384-wel plates.l A series of 1 O-fold dilution ofs the cell-targeting molecule to sbe teste wered prepared in an appropriate buffer, and 5 pL of the dilutions or buffer control wer eadded to the plated cells. Control wells containing only cel culturel medium wer usede for baseli necorrect ion.The cell samples wer e incubated with the cell-target molecing ules or just buffer for three or five days at 37°C and in an atmosphere of five perce ntcarbon dioxide (CO2). Ilie total cell survival or perce ntviability was determine usingd a luminesce readount usingt the CellTiter-Glo® Luminescent Cell Viability Assay (G7573, Promega Corp., Madison, WI, U.S.) according to the manufacturer’s instructions. 779. 779. 779. id="p-779" id="p-779" id="p-779" id="p-779" id="p-779" id="p-779" id="p-779" id="p-779" id="p-779" id="p-779"
[779] The Perce ntViabili tyof experimenta wellsl was calculated using tire foliovang equation: (Tes tRLU ״ Average Medi aRLU) / (Avera geCell onlys RLU - Average Media RLU) * 100. The logarithm of the cell-targeting molecule concentrat versionus Perce ntViabilit wasy plotted in Prism (GraphPad Prism San, Diego, CA, U.S.), and log (inhibitor) vers usrespons (3e parameter analysis) wasWO 2016/196344 PCT/US2016/034778 290 used to determine the half-maximal cytotoxic concentrat (CD50)ion value for die tested cell-target moleculing Thee. CD50 for eac hsample was calculated and is shown in Table 10. When CD50 value coulds not be calculat baseed don the shape of the curve over the concentra testetions thend, a. maximum CD50 value was noted as being beyond the maximum teste value,d e.g., great thaner 100 nM ("> 100.000 nM") or 200 nM ("> 200.000 nM"), for samples wrhich did not kill 50% of the cell s at the highest, tested, sample concentra e.g.,tion, 100.000 nM or 200.000 nM. If the cell viability in the assay was approxima tely50% at the highest test edsample concentra thention, tire CD50 value for that molecule was noted in Table 10 as being approximat theely maximum concentrati testeon whered the cell viability was approximat 50%,ely e.g., 100.000 nM." As reporte in dthe Examples, a. molecule exhibiting a CD50 within 10-fol ofd a CD50 exhibite byd a refere ncemolecule is considere to dexhibi cytot toxic activity comparable to that refere ncemolecule.
Table 10. Exempla ry,De-Immunize d,Protease-Cleavage Resista nt,Shiga. Toxin ______________ Effector Polypeptide Exhibiteds Potent Cytotocityxi Cell-Targeti ng Molecule CD5« (nM) CD50 (nM) scFv-1 target scFv-1 target Set 1 - scFv-1 positive cell line A positive cell line B Experime 1nt SLT-1A- 0.025 0.079 combo 10: :scFv-1 SLT-1A- 0.032 0.115 combo 16: :scFv-1 SLT-1A- 0.032 0.157 combo 19: :scFv-1 SLT-1A-FR:: scFv-1 0.014 0.045 Experime 2nt SLT-1A- 0.040 ~ 100.000 combo 17: :scFv-1 SLT-1A- > 100.000 > 100.000 combo 18: :scFv-1 SET-1A-FR:: scFv-1 0.007 0.048 SLT-1A-WT > 100.000 > 100.000 Experime 3nt SLT-1A- 0.026 combo? ::scFv-1 SET-1A-FR:: scFv-1 0.017WO 2016/196344 PCT/US2016/034778 291 Table 10 (cant’d) Cell-Targeti ng Molecule CD50 (nM) CD50 (nM) CD50 (nM) Set I, Experime 3nt scFv- 1targ et scFv-1 targe t scFv-1 target (cant’d) positiv cele linel F positive cell line G negative cel linel H SLT-1A- 0.386 0.133 > 100.000 combo? ::scFv-1 SLT-1A-FR: :scFv-1 0.283 0.099 > 100.000 scFv-1 target scFv-1 targe t Experime 4nt positive cell line A positive cell line G SLT-1A- 0.017 0.024 combo 1: :scFv-1 SLT-1A- 0.027 0.068 combo 10: :scFv-1 SLT-1A- 0.067 0.182 combo 12: :scFv-1 SLT-1A- 0.054 0.094 combo 15 ::scFv-1 SLT-1A- 0.088 0.162 combo 17: :scFv-1 SET-LVFR:: scFv-1 0.013 0.020 scFv- 1targ et scFv-1 targe t scFv-1 target Experime 5nt positive cell line A positive cel linel F positive cell line G SLT-1A- 0.01 0.12 0.02 combo? ::scFv-1 SLT-1A- 0.01 0.14 0.04 combo8: :scFv-1 SLT-1A- 0.01 0.33 0.13 combo9: :scFv-1 SLT-1A- 0.02 0.67 0.55 comboil ::scFv-1 scFv- 1targ et scFv-1 targe t scFv-1 target Experime 6nt positive cell line A positive cell line B positive cell line F SLT-1A- 0.03 0.25 0.84 combo 10: :scFv-1 SLT-1A- 0.17 0.79 3.21 combo20: :scFv-1WO 2016/196344 PCT/US2016/034778 292 Table 10. (cont’d) Cell-Targeti ng Molecule CD50 (nM) CD50 (nM) CD50 (nM) scFv-2 targ et scFv- 2targe t scFv- 2target Set 2 - scFv-2 positive cell line A positive cell line B positive cell line G Experime 7nt SLT-1A- 1.52 17.2 comboO:: scFv-2 SLT-1A- 0.68 3.18 combo3:: scFv-2 SLT-1A- 0.08 0.44 combo4::scFv2־ SLT-1A-FR:: scFv-2 0.01 0.06 Experiment 8 SLT-1A- 2.206 1.70 0.798 combo2:: scFv-2 SLT-1A- > 100.000 > 100.000 > 100.000 combo 13:: scFv-2 SLT-1A-FR:: scF v-2 0.119 0.171 0.112 Experime 9nt SLT-1A- 0.059 0.259 0.110 combo22:: scFv-2 SLT-1 A-FR: :scFv-2 0.041 0.172 0.070 scFv-3 scFv- 3target scFv- 3targe t scFv- 3target Experime 10nt positive cell line B positive cell line G negative cell line A. scFv-3:: SLT-1 A- 73.80 5.48 > 200.00 combo5 scFv-3:: SLT-1 A- 54.70 1.01 > 200.00 combo6 scFv-3:: SLT-1A 119.00 0.79 > 200.00 scFv-4 scFv-4 targe t scFv-4 targe t scFv-4 targe t Experime 11nt positive cel linel C positive cell line D negat ivecell line E SLT-1A- 0.045 0.059 > 200.000 combo? ::scFv-4 SLT-1A- 0.065 0.079 > 200.000 combo 14: :scFv-4 SLT- 1A-FR:: sc.Fv-4 0.012. 0.018 > 200.000WO 2016/196344 PCT/US2016/034778 293 Table 10. (cont’d) Cell-Targeti ng Molecule CD50 (nM) CD50 (nM) CD50 (nM) Set 3 - scFv-5 scFv- 5targe t scFv- 5targe t Experime 12nt positiv cee ll line I positive cell line H SLT-1A- 0.037 0.029 combo? ::scFv-5 SLT-lA-FR::scFv5 0.012 0.012 Experime 13nt SLT-1A- 0.064 comboS: :scFv-5 SLT-1A- 0.376 combo9:: scFv-5 SLT-1A- ~ 100.000 combo 11: :scFv-5 0.011 SLT-1A-FR:: scFv-5 scFv-6 target scFv-6 targe t Set 4 - scFv-6 positiv cee ll line I positive cell line H Experime 14nt SLT-1A- 0.037 0.157 combo? :sc: Fv-6 SLT-1A-FR:: scFv-6 0.075 0.038 Experime 15nt SLT-1A- 1.699 comboS: :scFv-6 SLT-1A- ~ 100.000 combo9: :scFv-6 SLT-1A-FR.:: sc.Fv-6 0.204 Experime 16nt SLT-1A- 0.240 combo? :sc: Fv-6 SLT-1A- 1.900 combo21: :scFv-6 SLT-1A- 0.260 combo23: :scFv-6 SLT-1A- 0.310 combo24:: scFv-6 SLT-1 A-FR::scFv-6 0.130WO 2016/196344 PCT/US2016/034778 294 Table 10. (cont’d) scFv-6 targ et Experime 17nt positiv cee ll line I SLT-1A- 0.500 combo? ::scFv-6 SLT-1 A- 0.100 combo25: :scFv-6 SLT-1 A- 0.730 combo26: :scFv-6 scFv- 8positive cel l scFv-8 negative scFv-8 Experime 18nt line B cell line A SLT-1A- 0.077 > 100.000 comboO: :scFv-8 SLT-1 A-FR: :scFv-8 0.440 > 100.000 780. 780. 780. id="p-780" id="p-780" id="p-780" id="p-780" id="p-780" id="p-780" id="p-780" id="p-780" id="p-780" id="p-780"
[780] The CD50 value fors exempla ry,SLT-1 A-combo scaffold-bas cell-ed, targeting molecules are shown in Table 10 and associated cell-kill assa datay is shown in Figure 3-10.s These results show the cell-kill assa resuy lts for at least thirty-f our,unique cell-, target moleculeing easch comprisi nga combination de- , immunized, Shiga toxin effector polypeptide SLT-1 A-combo(n) selected from at leas twenty-st Shigaix toxin effector polypepti des(SEQ ID NOs: 6-27 and 29-32).
Thirty-two of thirty-four combination, de-immunized, Shiga toxin effector polypepti desSLT-lA-combo(0-12, 14-17, 19-21, 23-27, and 29-32) effectuate d cytotoxicit in ies,the context of a cell-targeti molecng ule, characteriz by edCD50 values equa tol or les thans 100 nM, wherea twos cell-target moleculesing , comprising either SLT-lA-combol3 (SEQ ID NO:19) and SLT-lA-combol8 (SEQ ID NO :24), exhibite cytotoxicd charaities cteriz by edCD50 values great thaner 100 nM towar thed two cell-lines tested (Tabl e10; Figures 6-7). 781. 781. 781. id="p-781" id="p-781" id="p-781" id="p-781" id="p-781" id="p-781" id="p-781" id="p-781" id="p-781" id="p-781"
[781] The results reporte in dTable 10 show that the cell-target moleculesing comprising SLT-1 A-comboO, SLT-1 A-combo 1, SLT-1 A-combo4, SLT-1A- combo?, SLT-1 A-combo8, SLT-1 A-combo9, SLT-1 A-combo 10, SLT-1 A- comboil, SLT-lA-combol2, SLT-lA-combol4 SLT-lA-, combol5 SLT-1A-, combol6, SLT-lA-combol7, SLT-lA-combol9 or ,SLT-lA-combo25 displayed potent cytotoxic characities ter byized CD50 value ofs 0.2 nM or less depending on the cell-line teste (Tabled 10; Figures 3-10). The cytotoxici ofties most of the Shiga toxin effector polypepti descombo(n) teste asd a component of a cell-targetin g molecule wer ecomparable to the cytotoxici ofties a SLT-1 A-FR polypeptide (SEQID NO:5) as a component of a relate celd, l-target molingecule (Tabl e10; Figures 3­ 7; 9-10). For example, the cytotoxiciti to ates least one cell-type tested for at leas t one cell-targeti molngecule comprisin oneg of the Shig atoxin effector polypepti des combo(0-2, 4, 7-8, 10, 12, 14-19, or 23-26) were compara toble the cytotoxici ties of relate celd, l-targeting molecules comprising a SLT-1A-FR polypept ide(SEQ ID NO:5) (Table 10; Figures 3-7; 9-10). 782. 782. 782. id="p-782" id="p-782" id="p-782" id="p-782" id="p-782" id="p-782" id="p-782" id="p-782" id="p-782" id="p-782"
[782] The specifici ofty the cytotoxiciti of exemplary,es cell-targeti molengcules comprising certain combina, tion, de-immunized, Shiga toxi effecn tor polypeptides SLT-1 A-combo(n) were determine usingd target-negative cell-kil asslays known to the skill edworke Ther. target biomolecu negatle ivecell-kill assay is identical to the target-posit cell-ive kill assay except for the cel typel used. The target negat ivecell­ kill assay was perform usinged cell whichs do not express significant amounts of the extracell targetular biomolecule bound with high-affinit by they respectiv bindinge region scFv-(n) of the cell-targeti molngecul beinge tested. The cytotoxi c specificit ofies exemplary, cell-target moleingcules comprising certai combn, inatio n, de-immunized, Shiga toxin effector polypept idescaffol SLT-1ds A-combo(n) wer e determined by comparing the results from target positive cell-kill assays wit hresults from target-negati cell-kve assillays (see e.g. Table 10; Figures 8-9). Cytotoxic, cell-targeti molengcules comprising de-immunize Shigd, atoxi effectorn polypepti desSLTl-A-combo(5-7, 14, or 21) or SLT-1 A-FR (SEQ ID NO:5) did not kill comparab percentle ages of target negat ivecell ass compared to target positive cell ats the same, cell-targeti molngecul concentratie (Tablons e10; Figures 8-9).
C. Testing for Caspase Activation Induced by Exemplary, Cell-Targeti Molecng ules Comprising Shiga Toxin Effector Polypepti desSLT-1 A-combo(n) 783. 783. 783. id="p-783" id="p-783" id="p-783" id="p-783" id="p-783" id="p-783" id="p-783" id="p-783" id="p-783" id="p-783"
[783] Apoptosis is a programm celed deatl involvingh the degradati ofon cellu lar components by caspases. Apoptosis can be detected by monitoring the activ ityof the effector caspases 3 and 7, such as, e.g., the activat station ofe either caspase 3 or caspase 7. The caspase activation of exemplary, cell-targeti moleculng es comprising combinatio de-immn, unized, protease-cleavage resistant, Shiga toxi n effect polypepor tid SLT-1es A-combo(n) of the present invention were determined using a target biomolec ulepositive cell caspase, activ ityassa knowny to the skilled worker The. method of the target positive cell, caspase activity assa isy analogous to the target-posit cellive-kil assayl described above. -295-WO 2016/196344 PCT/US2016/034778 296 784. 784. 784. id="p-784" id="p-784" id="p-784" id="p-784" id="p-784" id="p-784" id="p-784" id="p-784" id="p-784" id="p-784"
[784] Shiga toxin catalytic activity can induc eapoptosis of Shiga toxin-intoxicat ed, mammalian cells via an intrinsic pathway (see e.g.. Inward C et al., J Infect 30: 213- 8 (1995); Fujii J et al., Infect Immun 71: 2724-35 (2003); Jandhyala D et al., Curr Top Microbiol Immunol 357: 41-65 (2012); Tesh V, Curr Top Microbiol Immunol 357: 137-78 (2012)). Caspas acte ivati canon be used as a readou fort the activat ion of apoptoti mechanic sms leading to cell deat (seeh Pop C, Salvesen G, J Biol Chern 284: 21777-81 (2009); Nichol S,ls Hyman B, Methods Enzymol 544: 251-69 (2014)). The potenc andy specific ityof caspas acte ivati inducedon by the combination de-immunized,, protease-cleava resistage Shigant, toxin effector polypepti desSLT-1 A-combo7 and SLT-lA-combol4 in the context of a cell- targeting molecules wer edetermined as follows. 785. 785. 785. id="p-785" id="p-785" id="p-785" id="p-785" id="p-785" id="p-785" id="p-785" id="p-785" id="p-785" id="p-785"
[785] The caspase activati ofon cell-targeti molecng ules comprisin de-g immunized, protease-cleava resistage Shigant, toxin effecto polypeptider scaffolds wer edetermined using cells expressi ng,at a cellular surface, significant amounts of an extracellular targe biomolecult of ethe binding region scFv- 1or scFv-6. 786. 786. 786. id="p-786" id="p-786" id="p-786" id="p-786" id="p-786" id="p-786" id="p-786" id="p-786" id="p-786" id="p-786"
[786] The caspase activati assonay was performed as follows. Certain human, tumor, cell-line cells wer platede (at about 2-8 x 103 cells per wel l)in 20 microliter (pL) cel culturel medium in 384-well plates. A serie ofs 10-fold dilutions of the cell-targetin molecg ules to be teste wered prepared in an appropriate buffe r,and 5 pL of the dilution ors buffer control were added to the plate celld s.Control wells containing only cell culture medium wer usee d for baseline correcti Theon. cel l samples wer eincubated with the cell-target moleculesing or just buffer for 18-20 hour ats 37°C and in an atmosphe ofre five percent CO2. The caspase activation was determined using a luminesce readoutnt using the Caspase-Gio 3H® Luminescent Cell Viabili tyAssa y(Promega Corp., Madison, WI, U.S.) according to the manufactur’s inster ructions. 787. 787. 787. id="p-787" id="p-787" id="p-787" id="p-787" id="p-787" id="p-787" id="p-787" id="p-787" id="p-787" id="p-787"
[787] The amount of caspase activation in experimental well wass calculated using the followi equation:ng ((Test RLU - Avera geMedia RLU) / (Avera geCell s RLU - Average Media RLU)). * 100. Hie logarit ofhm the cell-target molecing ule concentrat versionus Caspase Activati wason plotted in Prism (GraphPad Prism San, Diego, CA, U.S.) and log (agonist) vers usrespons (3e paramet analer) ysis was used for each, tested, cell-targetin moleculeg to calcul atethe half-maxima effel ctive concentration (EC50) value for caspas active ation in tire assa (Figurey s11-12; Table 11). Hie maximum caspas actie vity percenta (thege perce ntactivati overon theWO 2016/196344 PCT/US2016/034778 297 ‘־cells only" contr measol urement fors) eac hexperiment (maximum activity was) calculat usinged caspase activation measurements from samples with "cells only" as the baseline (Table 11). The EC50 and maximum activit fory caspase activat forion exemplar cell-y target molecing ules are shown in Table 11.
Table 11. Exempla ry,De-immunized, Protease-Clea Resistvage ant, Cell-Targeting Molecules Induced Caspas Acte ivation caspa se maximum caspa se Cell-Targeti ng maximum activity ECso activit y activ ityEC50 Molecule (nM) (nM) activ ity(%) (%) scFv-1 targe positit ve cell scFv-1 targe positt ive cell line A line G SLT-1A- 0.072 562 0.066 245 00mb07::scFv-l SLT-1A- 0.083 534 0.094 245 combol4::scFv-l SLT-1A- 0.054 561 0.045 250 FR::scFv-l scFv- 7targe positit ve cell scFv-7 targe posit tive cell line H line I SLT-1A- 2.410 219 0.210 216 combo7:: scFv-7 SLT-1A- 0.860 335 0.050 234 FR:: scFv-7 788. 788. 788. id="p-788" id="p-788" id="p-788" id="p-788" id="p-788" id="p-788" id="p-788" id="p-788" id="p-788" id="p-788"
[788] The caspase activity in targe cellst induced by the Shiga toxin effector polypept idecombos 7 and 14, eac htested as a component of a cell-targeting molecule, wer ecomparable to the caspase activity induced by the SLT-1A-FR polypept ide(SEQ ID NO:5) as a component of a relat ed,cell-targetin moleculeg (SEQ ID NO:34 and SEQ ID NO:39, respective forly) most of the cell-line testes d (Tabl e11; Figures 11-12).
D. Endogenous Epitope Disrupting Mutations in Shiga Toxin Effector Polypeptides 789. 789. 789. id="p-789" id="p-789" id="p-789" id="p-789" id="p-789" id="p-789" id="p-789" id="p-789" id="p-789" id="p-789"
[789] This Example show's that Shiga toxin A Subunit effector polypeptides can be de-immunized with certain truncatio andns combinations of amino acid residue substitutions. Deletions and/or amino acid substitutions wer emade in the putativ e, B-cell and/o T-cer ll epitopes of Shiga toxin effector polypepti desderived from the A Subunit of Shiga-like Toxin 1 (SLT-1A) as liste ind Table 12. In this Examples and in WO 2015/113005, many mutations have been empirical testely ford effect(s on) the Shiga toxin effecto functir onof various Shiga toxin effector polypepti desand cell-target molecules.ing Table 12 summarizes the results describe ind theWO 2016/196344 PCT/US2016/034778 298 Examples and in WO 2015/113005 where an amino acid substitut ionor combinat ion of amino acid substitutions did not prevent the exhibition of a potent leve ofl Shiga toxin effector function. 'Table 12 uses the epitope region numbering scheme describe ind the Exampl 1e (see Table 7, supra), and lists any change in B-cell epitopes predicted by BcePre softwd are.
Table 12. Substitutions and Combinations of Substitutions Empiricall Veriy fie d Not to Prevent Exhibition of a Potent Shiga Toxin Effector Function(s) natively positioned amino aci dpositions Epitope Ribosome Epitope Region Substitution(s) PredictionInhibitionCytotoxicity K1M/K1 j A no change YES YES 1 1 S8I no change YES YES 1 T9I no change YES YES 2 S331 eliminated YES YES 1 3 T45I eliminated YES YES 3 S45I eliminatedYES YES 4 D53A no change YES YES 4 R55A no change YES YES 4 D58A eliminatedYES YES 4 D58F eliminated YES YES 4 P59A eliminatedYES YES | 4 E60I eliminatedYES YES 4 E60R no change YES YES 4 E61A eliminated YES YES 4 G62A eliminated YES YES D94A/S96I no change YES YES 7 DI 83 A no change YES YES 7 D184A eliminatedYES YES D184F eliminated YES YES י R188A eliminatedYES YES י 7 D183A/D184A/ eliminatedYES YES R188A immunogeni c R205A eliminated YES YES | residue | 4 and 5 E60I/ G110A eliminatedYES YES 1 4 and 6 E60I/ G147A eliminated YES YES | 3 and 7 T45I/R188A eliminatedYES YES 1 3 and 7 S45I/R188A eliminatedYES YES | 2, 4, and 6 S33I/ G110A/ eliminated YES YES G147A/ pun ،9 ،c > i-ti i-ti i-ti i-ti 3? A ° oo || oc ؛ Q O WO 2016/196344 PCT/US2016/034778 299 T45IZ 3, 4, and 6 eliminated YES YES G110A/G147A | 3, 4, and 6 S45IZ eliminated YES YES G110A/G147A | 4, 5, and 6 D58AZG110AZ eliminated YES G147A E60IZ G110AZ 4, 5, and 6 eliminated YES YES G147A T45IZ D58AZ E60IZ 3, 4, 5, and 6 eliminated YES YES G 110 AZ G 147 A | 3, 4, 5, and 6 S45IZ D58AZ E60IZ eliminated YES YES G110AZ G147A | 3, 4, 5, and 6 T45IZ D58AZ E60IZ G62AZ G110AZ eliminated YES YES G147A | 3, 4, 5, and 6 S45IZ D58AZ E60IZ G62AZ G 11OAZ eliminated YES YES G147A T45IZGH0AZ 3, 4, 5, 6, and 7 G 147AZ DI 83AZ eliminated YES YES D184AZR188A S45IZ G110AZ 3, 4, 5, 6, and 7 G147AZ D183AZ eliminated YES YES D184AZR188A 4, 5, 6, and 7 eliminated YES D58AZ G110AZ 4, 5, 6, and 7 eliminated YES YES G147AZ G187A D58A/ G110AZ 4, 5, 6, and 7 eliminated YES YES G147AZR188A D58AZG110AZ eliminated YES YES G147AZ S189A | 4, 5, 6, and 7 D58A/G110AZ G147A/S186AZ eliminated YES YES R188A D58A/G110AZ 4, 5, 6, and 7 G147AZ G187AZ eliminated YES R188A S33IZ S45IZ D58AZ 2, 3, 4, 5, and 6 eliminated YES YES G 11 OAZ G 147 A T45IZ D58AZ E60V G110AZ G147AZ 3, 4, 5, 6, and 7 eliminated YES YES D183A/D184AZ R188A | 3, 4, 5, 6, and 7 S45IZ D58AZ E60IZ eliminated YES G110AZ G147AZ ؛WO 2016/196344 PCT/US2016/034778 300 D183AZD184AZ R188A T45IZ D58A/ E60I/ G62AZ G110A/ 3, 4, 5, 6, and 7 eliminatedYES YES G147A/D183A/ D184A/R188A S451/ D58A/ E601/ G62A/G110AZ 3, 4, 5, 6, and 7 eliminatedYES YES G147A/ D183AZ D184A/R188A D58AZ G110AZ G147A/R188AZ 4, 5, 6. 7, and 8 eliminatedYES YES C242S/ R248AZ R251A T104N/A105LZ T107P/L108MZ and 8 S109V/G110AZ eliminatedYES YES D11rr/R248A/ R251A V54IZ R55L/I57FZ P59FZ E60TZ L6 H. 4 and 8 eliminated YES R248AZR251A S43N/ G44LZ T45V/ G46PZ D47MZ N48VZ 3 and 8 eliminatedYES YES L49A/ F50TZ A5IV/ R248A/ R251A S43N/ G44LZ S45VZ G46PZ D47M/N48V/ 3 and 8 eliminated YES YES L49A/ F50TZ A51V/R248AZ R251A T45I/V54I/R55LZ 157F/ P59F/ E60TZ 3, 4, and 8 eliminated YES YES E61LZ C242SZ R248A/R251A S45I/ \ 54L R55L/ I57FZ P59FZ E60TZ 3, 4, and 8 eliminatedYES YES E61L/ C242SZ R248A/R251A D53N/ V54L/ R55VZ G56PZ I57M/ D58V/ 4 and 8 eliminatedYES YES P59A/ E60T7 E61VZ R248A /R251AWO 2016/196344 PCT/US2016/034778 301 T45IZ V54IZ R55LZ 157F/ P59F/ E60T/ 3, 4, 5, and 7 eliminated YES E61L/G110AZ R188A S45I/ V54I/R55L/ I57FZ P59FZ E60TZ 3, 4, 5, and 7 eliminated YES E61LZ G110AZ R188A D58AZ G110A/ 4, 5, 6, and 8 G147A/R188AZ eliminatedYES YES C242S T4517 ¥541/R55LZ 7FZ P59FZ E60T/ 3, 4, 5, 6, and 7 eliminated YES E61L/GH0A/ G147A/ R188A S45TZ V54IZ R55LZ I57FZ P59FZ E60T/ 3, 4, 5, 6, and 7 eliminated YES E61L/G110AZ G147A/ R188A T45IZ V54IZ R55LZ I57FZ P59FZ E60TZ 3, 4, 5, 7, and 8 E61L/G110A/ eliminated YES R188AZ C242SZ R248A/R251A S45IZ ¥541/ R55L/ 7F/ P59FZ E60TZ 3, 4, 5, 7, and 8 E61L/G110A/ eliminated YES R188A/C242SZ R248A/R251A 3, 4, 5, 7, and 8 T45I/V54I/R55LZ 7F/ P59FZ E60TZ E61LZ R84AZ eliminated YES G110AZ R188AZ C242S/ R248AZ R251A 3, 4, 5, 7, and 8 S451/ ¥541/ R55LZ I57F/P59FZ E60T/ E61L/R84AZ eliminated YES G110AZR188AZ C242S/ R248AZ R251A 3, 4, 5, 7, and 8 T45I/V54I/R55LZ 7F/ P59FZ E60TZ E61L/V88A/ eliminated YES G110AZ R188AZ C242SZ R248AZ R251A 3, 4, 5, 7, and 8 S451/ ¥541/ R55LZ eliminated YES I57F/P59FZ E60T/WO 2016/196344 PCT/US2016/034778 302 E61LZ ¥8 8 AZ G110A/R188AZ C242SZ R248AZ R251A 3, 4, 5, 7, and 8 T45IZ V54IZ R55LZ I57FZ P59FZ E60TZ E61L7 G110AZ eliminated YES D141A/R188AZ C242SZ R248AZ R251A 3, 4, 5, 7, and 8 S45TZ V54IZ R55LZ I57FZ P59FZ E60T7 E61LZG110AZ eliminated YES D141 AZ R188AZ C242SZ R248AZ R251A 3, 4, 5, 7, and 8 T45IZ V54IZ R55LZ I57FZ P59FZ E60TZ E61L7 G110 AZ eliminated YES V154A/R188AZ C242SZ R248AZ R251A 3, 4, 5, 7, and 8 S45TZ V54IZ R55LZ I57FZ P59FZ E60TZ E61LZG110AZ eliminated YES V154AZ R188AZ C242SZ R248AZ R251A 3, 4, 5, 7, and 8 T45I/V54IZR55LZ I57FZ P59FZ E60TZ E61LZ G110 AZ eliminated YES R188AZ D198AZ C242SZ R248AZ R251A 3, 4, 5, 7, and 8 S45TZ V54IZ R55LZ I57FZ P59FZ E60TZ E61L/G110AZ eliminated YES R188AZ D198AZ C242SZ R248AZ R251A T4517 ¥541/R55LZ I57FZ D58VZ P59FZ E60TZ E61LZ 3, 4, 5, 7, and 8 eliminated YES G110A/R188AZ C242SZ R248A/ R251A S45I/ ¥5417 R55LZ 3, 4, 5, 7, and 8 I57FZ D58VZ P59FZ eliminated YES E60TZ E61L/i-ti WO 2016/196344 PCT/US2016/034778 303 G110A/R188AZ C242SZ R248A/ R251A T45IZ D53GZ V54IZ R55LZI57FZ P59FZ E60TZ E61LZ 3, 4, 5, 7, and 8 eliminated YES G110AZR188AZ C242SZ R248AZ R251A S45I/D53GZ V54IZ R55LZI57FZ P59FZ E60T7 E61LZ 3, 4, 5, 7, and 8 eliminated G110A/R188AZ C242SZ R248AZ R251A T45IZV54TZR55LZ I57FZ P59FZ E60TZ 3, 4, 5, 6, and 8 E61LZG110AZ eliminated YES YES G 147 AZ C242S/ R248AZR251A S45IZ V54IZ R55LZ I57FZ P59FZ E60TZ 3, 4, 5, 6, and 8 E61L/GH0AZ eliminated YES YES G 147 AZ C242S/ R248AZR251A T45IZ D53GZ V541Z R55L/ I57FZ D58VZ P59FZ E60TZ L6 H. 3, 4, 5, 7, and 8 eliminated YES G110A/R188AZ C242SZ R248AZ R251A S45IZ D53GZ V54IZ R55LZI57FZ D58VZ P59FZ E60TZ E61LZ 3, 4, 5, 7, and 8 eliminated YES G110AZ R188AZ C242SZ R248AZ R251A K1AZ S45IZ V54IZ R55LZI57F/P59FZ E60TZ E61LZ 3, 4, 7, and 8 eliminated YES G110A/G147AZ C242SZ R248AZ R251A S45IZ V54IZ R55L/ I57FZ P59FZ E60TZ E61LZ T180GZ 3, 4, 7, and 8 eliminated YES T181VD183GZ D184FZ L185DZ S186F/G187TZWO 2016/196344 PCT/US2016/034778 304 C242SZ R248AZ R251A T45IZV54IZR55LZ 7F/ P59FZ E60TZ E61LZGL10AZ 3, 4, 5, 6, 7, and 8 eliminated YES G147AZR188AZ C242SZ R248AZ R251A S45IZ V54IZ R55LZ I57FZ P59FZ E60TZ E61L/GH0AZ 3, 4, 5, 6, 7, and 8 eliminated YES G147AZR188AZ C242SZ R248AZ R251A K1A/T45IZ V54IZ R55LZ T57FZ P59FZ E60TZ E61LZ 1, 3, 4, 5, 6, 7, and 8 eliminated YES G110AZ G147AZ R188AZC242SZ R248A/R251A K1AZ S45IZ V54IZ R55LZI57FZ P59FZ E60TZ E61LZ 1, 3, 4, 5, 6, 7, and 8 eliminated YES G110A/G147AZ R188AZ C242SZ R248AZR251A T4IZ T45IZ V54IZ R55LZI57FZ P59FZ E60TZ E61LZ 1, 3, 4, 5, 6, 7, and 8 eliminatedYES YES G110AZ G147AZ R188A/C242SZ R248A/R251A T4IZ S45IZ V541/ R55LZI57FZ P59FZ E60TZ E61LZ 1, 3, 4, 5, 6, 7, and 8 eliminatedYES YES G110A/G147AZ R188AZ C242SZ R248AZR251A K1AZ T4IZ T45IZ V54IZ R55LZI57FZ P59F/ E60TZ E61LZ 1, 3, 4, 5, 6, 7, and 8 eliminated YES G110AZ G147AZ R188AZ C242SZ R248A/R251A KI AZ T4IZ S45IZ V54IZ R55LZI57FZ 1, 3, 4, 5, 6, 7, and 8 eliminated YES P59FZ E60TZ E61LZ G110A/G147AZWO 2016/196344 PCT/US2016/034778 305 R188AZ C242S/ R248A/R251A T4IZ T45IZ D58AZ E60IZ G62AZ G110A/G147AZ 1, 3, 4, 5, 6, 7, and 8 eliminated YES YES D183A/D184AZ R188A/C242SZ R248A/R251A T4I/ S45I/ D58AZ E60IZ G62AZ G110AZ G147AZ 1, 3, 4, 5, 6, 7, and 8 eliminatedYES YES D183A/D184AZ R188AZ C242SZ R248A/R251A T45IZ V54IZ R55LZ I57FZ P59FZ E60TZ E61LZG110AZ 3, 4, 5, 6, and 8 eliminated YES G147AZ Vins 11 residues/ C242S/ R248A/R251A S45IZ V54I/ R55LZ I57FZ P59FZ E60TZ E6 IL/GUO A/ 3, 4, 5, 6, and 8 eliminated YES G147AZ Vins 11 residues/ C242SZ R248A/R251A 790. 790. 790. id="p-790" id="p-790" id="p-790" id="p-790" id="p-790" id="p-790" id="p-790" id="p-790" id="p-790" id="p-790"
[790] In epitope regions 1-5 and 7-8, different amino acid substitutions have been made and test ed(see Table 12). In epitope region #1 (see Table 7), the lysine natively located at position 1 in the matur Ae Subunit sof Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alani ne(KIA) and methionine (K IM)). In epitope region #1, the threonine native locatly ated position 4 in the mature A Subunit sof Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to isoleucine (T4I). In epitope regio #1,n the asparta te natively located at position 6 in the matur Ae Subunit sof Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to arginine (D6R). In epitope region #1, the serine natively located at position 8 in the mature A Subunit s of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutat ed to isoleucine (SSI). In epitope region #1, the threoni nativelyne located at position 9 in the mature A Subunit sof Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) wras mutated to isoleucine (T9I) and to valine (T9V). In epitope region #1, the lysine natively locat ated position 11 in the matur Ae Subunits ofWO 2016/196344 PCT/US2016/034778 306 Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (KI LA) and to histidine (KI 1H). In epitope region #1, the threonine natively located at position 12 in the mature A Subunits of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to lysine (T12K). 791. 791. 791. id="p-791" id="p-791" id="p-791" id="p-791" id="p-791" id="p-791" id="p-791" id="p-791" id="p-791" id="p-791"
[791] In epitope region #2 (see Table 7), the serine natively located at position 33 m the matur Ae Subunit sof Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutat edto isoleucine (S33I). 792. 792. 792. id="p-792" id="p-792" id="p-792" id="p-792" id="p-792" id="p-792" id="p-792" id="p-792" id="p-792" id="p-792"
[792] In epitope region #3 (see Table 7), the serine native localy ted at position 43 in tiie mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) was mutated to asparagine (S43N). In epitope regio #3,n the glycine native locatly ated position 44 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) was mutat edto leucine (G44L). In epitope region #3, the serine natively located at position 45 in the matur Ae Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) was mutat edto valine (S45V) and to isoleucine (S45I). In epitope region #3, the threon nativeine locatedly at position 45 in the matur Ae Subunit of Shiga-like toxin 1 (SEQ ID NO: 2) was mutated to valine (T45V) and to isoleucine (T45I). In epitope region #3, the glycine natively located at position 46 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) was mutated to proline (G46P). In epitope regio #3,n the asparta te natively located at position 47 in the matur Ae Subuni tof Shiga-like toxin 1 (SEQ ID NO: I) was mutated to glycine (D47G) and to methionine (D47M). In epitope region #3, the asparagine natively located at position 48 in the matur Ae Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) was mutat edto valine (N48V) and to phenylala (N48F).nine 793. 793. 793. id="p-793" id="p-793" id="p-793" id="p-793" id="p-793" id="p-793" id="p-793" id="p-793" id="p-793" id="p-793"
[793] In epitope region #4 (,see Table 7), the aspartate natively located at position 53 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (D53A), glycine (D53G), and asparagine (D53N). The D53 residue was predicted by the Epitopia ■webserver to be solvent exposed and have an immunogenicit scayle value of 5 or "high." In epitope region #4, the valine natively located at position 54 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: I) and Shiga toxin (SEQ ID NO:2) was mutated to isoleucine (V54I). In epitope region «4, the arginin nativelye located at position 55 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (R55A), to valine (R55V), and to leucine (R55L). In epitope region #4, the glycine native localy ted at position 56 in the mature AWO 2016/196344 PCT/US2016/034778 307 Subuni tof Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to proli ne(G56P). In epitope region #4, the isoleucine native localy ted at position 57 in the matur Ae Subuni tof Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to methionine (D57M) and to phenylala nine (D57F). In epitope regio #4,n the aspartate native localy ted at position 58 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutat edto alanine (D58A), to valine (D58V), and to phenylala nine (D58F). In epitope region M, the proline natively located at position 59 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (P59A). In epitope region #4, the glutamate natively located at position 60 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to isoleucine (E60I), to threoni ne (E60T), and to arginin (E60R).e In epitope regio #4,n the glutama nativelyte located at position 61 in the matur Ae Subuni tof Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (E61 A), to valine (E61V), and to leucine (E61L). In epitope regio #4,n the glycine native localy ted at position 62 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (G62A). 794. 794. 794. id="p-794" id="p-794" id="p-794" id="p-794" id="p-794" id="p-794" id="p-794" id="p-794" id="p-794" id="p-794"
[794] In epitope region #5 (see Table 7), the aspartate natively located at position 94 m the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutat edto alanine (D94A). In epitope region #5, the serine natively located at position 96 in the matur Ae Subunit of Shiga-like toxi n1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to isoleucine (S96I). In epitope region #5, the threonine natively located at position 104 in the mature A Subunit of Shiga-like toxi n1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to asparagine (T104N). In epitope regio #5,n the alani nenatively located at position 105 in tire matur Ae Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID \O 2) was mutated to leucine (A105L). In epitope region #5, the threon nativeine locately atd position 107 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to proline (T107P). In epitope region #5, the leucine natively locat ated position 108 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to methionine (L108M). In epitope region #5, the serine natively located at position 109 in the mature A Subunit of Shiga-like toxin 1 (SEQ WO 2016/196344 PCT/US2016/034778 308 ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to valine (S109V). In epitope region #5, the glycine natively located at position 110 in the matur Ae Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (G110A). In epitope regio #5,n the aspartate native locatly ated position 111 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to threonine (Dll IT). In epitope regio n #5, the serine natively located at position 112 in the matur A.e Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to valine (SI 12V). 795. 795. 795. id="p-795" id="p-795" id="p-795" id="p-795" id="p-795" id="p-795" id="p-795" id="p-795" id="p-795" id="p-795"
[795] In epitope regio #6n (see Table 7), the aspartate natively located at position 141 in the matur Ae Subunit of Shiga-li ketoxin 1 (SEQ ID NO: 1) and Shiga toxin, (SEQ ID NO:2) was mutated to alanine (D141A). In epitope region #6, the glycine natively located at position 147 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to alani ne(G147A). 796. 796. 796. id="p-796" id="p-796" id="p-796" id="p-796" id="p-796" id="p-796" id="p-796" id="p-796" id="p-796" id="p-796"
[796] In epitope region #7 (see Table 7), the arginine natively located at position 179 in die matur Ae Subuni tof Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (R179A). This R179 residue was predicted by the Epitopia webserver to be expose andd have an immunogenicit valuey of 5 or "high." In epitope region #7 (see Table 7), the threonine natively located at position 180 m the mature A Subunit of Shiga-like toxin I (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutat edto glycine (T180G). In epitope region #7 (see Table 7), the threon nativeine localy ted at position 181 in the matur Ae Subunit of Shiga - like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to isoleucine (T18II). In epitope regio #7n (see Table 7), the asparta natite vely located at position 183 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (D183A) and to glycine (D183G). In epitope regio #7,n the aspartate native locatedly at position 184 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (D184A) or phenylala (D184Fnine ). In epitope regio #7n (see Table 7), the leucine natively located at position 185 in the matur Ae Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO :2) was mutated to valine (LI85V) or aspartate (LI 85D). In epitope region #7, the serine natively located at position 186 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alani ne(S186A) and to WO 2016/196344 PCT/US2016/034778 309 phenylala (S186F).nine In epitope region #7, the glycine natively located at position 187 in the mature A Subunit of Shiga-like toxin I (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutat edto alanine (G187A) and to threonine (G187T). In epitope region #7, the arginin nativelye located at position 188 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (R188A) and to leucine (RI88L). In epitope regio n #7, the serine natively located at position 189 in the matur A.e Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alani ne (S189A). 797. 797. 797. id="p-797" id="p-797" id="p-797" id="p-797" id="p-797" id="p-797" id="p-797" id="p-797" id="p-797" id="p-797"
[797] In epitope regio #8n (see Table 7), the arginine natively located at position 248 in the matur Ae Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin, (SEQ ID NO:2) was mutated to alanine (R248A). In epitope region #8, the arginine natively located at position 251 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to alani ne(R251A). 798. 798. 798. id="p-798" id="p-798" id="p-798" id="p-798" id="p-798" id="p-798" id="p-798" id="p-798" id="p-798" id="p-798"
[798] The leucine natively locate atd position 49 in the mature A Subunit of Shiga - like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (L49A). This L49 residue was predicted by the Epitopia webserver to be solvent expose andd have an immunogenicit valuey of 4 and is prese ntin T-cell epitope #2 (see Table 7). The glutama natite vely located at position 198 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutat edto alanine (D198A). This D198 residue was predicted by the Epitop ia webserver to be solvent expose andd have an immunogenicit valuey of 5 or "high." The arginine natively located at position 205 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (R205A). This R205 residue was predicted by the Epitopia webserver to be solvent expose andd have an immunogenicit valuey of 5 or "high." 799. 799. 799. id="p-799" id="p-799" id="p-799" id="p-799" id="p-799" id="p-799" id="p-799" id="p-799" id="p-799" id="p-799"
[799] In T-cell epitop es#1 through #6, differ entamino acid substitutions have been made and tested (see Table 12). 800. 800. 800. id="p-800" id="p-800" id="p-800" id="p-800" id="p-800" id="p-800" id="p-800" id="p-800" id="p-800" id="p-800"
[800] In T-cell epitope #1 (see Table 7), the threonine native localy ted at position 4 in die mature A Subunit sof Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to isoleucine (T4I), the aspartate native localy ted at position 6 in the mature A Subunits of Shiga-hke toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to arginine (D6R), die serine nativelv located at position 8 m the mature A Subunits of Shiga-hke toxin 1 (SEQ ID NO: 1) and Shiga WO 2016/196344 PCT/US2016/034778 310 toxin (SEQ ID NO:2) was mutated to isoleucine (SSI), the threonine natively located at position 9 in the mature A Subunits of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutat edto isoleucine (T9I) and to valine (T9V), the lysine natively located at position 11 in the matur Ae Subunits of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutat edto alani ne(KI 1A) and to histidine (KI 1H), the threon natiine vely located at position 12 in the mature A Subunits of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to lysine (T12K), and the serin nativee localy ted at position 33 in the mature A Subunits of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutat edto isoleucine (S33I). 801. 801. 801. id="p-801" id="p-801" id="p-801" id="p-801" id="p-801" id="p-801" id="p-801" id="p-801" id="p-801" id="p-801"
[801] In T-cell epitope #2 (see Table 7), the serine natively located at position 43 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) was mutated to asparagine (S43N); tire glycine natively located at position 44 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) was mutated to leucine (G44L); the serine natively located at position 45 in the matur Ae Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) was mutated to valine (S45V) and to isoleucine (S45I); the glycine natively located at position 46 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) was mutated to proline (G46P); the aspartate natively located at position 47 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) was mutated to glycine (D47G) and to methionine (D47M); the asparagine natively located at position 48 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) was mutated to valine (N48V) and to phenylala (N48F);nine the phenylalanine natively located at position 50 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (F50T); the alanine natively located at position 51 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to valine (A51V); the aspartate natively located at position 53 in the matur Ae Subunit of Shiga-like toxin 1 (SEQ ID NO:I) and Shiga toxin (SEQ ID NO:2) was mutated to alani ne(D53A), glycine (D53G), and asparagine (D53N); the valine native localy ted at position 56 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to leucine (V54L); the arginin nativelye located at position 55 in the matur Ae Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (R55A), to valine (R55V), and to leucine (R55L); the glycine natively located at position 56 in tire matur Ae Subunit of Shiga-like toxin 1 WO 2016/196344 PCT/US2016/034778 311 (SEQ ID NO; 1) and Shiga toxin (SEQ ID NO:2) was mutat edto proline (G56P); the isoleucine natively located at position 57 in the matur Ae Subuni tof Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to methionine (D57M) and to phenylala (D57F)nine ; the aspartate native localy ted at position 58 in the matur Ae Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (D58A), to valine (D58V), and to phenylala nine (D58F); the proline natively located at position 59 in the mature A. Subunit of Shiga - like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (P59A); the glutama nativete localy ted at position 60 in the matur Ae Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to isoleucine (E60I), to threonine (E60T), and to argmine (E60R); the glutamat e natively located at position 61 in the matur Ae Subuni tof Shiga-like toxin I (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to alani ne(E61A), to valine (E61V), and to leucine (E61L); the glycine natively located at position 62 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (G62A). 802. 802. 802. id="p-802" id="p-802" id="p-802" id="p-802" id="p-802" id="p-802" id="p-802" id="p-802" id="p-802" id="p-802"
[802] In T-cell epitope #3 (see Table 7), the aspartate natively located at position 94 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (D94A), and the serine natively located at position 96 in the mature A Subunit of Shiga-like toxin I (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutat edto isoleucine (S96I). 803. 803. 803. id="p-803" id="p-803" id="p-803" id="p-803" id="p-803" id="p-803" id="p-803" id="p-803" id="p-803" id="p-803"
[803] In T-cel epitl ope #4 (see Table 7), the glycine natively located at position 147 in die matur Ae Subuni tof Shiga-like toxin I (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (G147A). 804. 804. 804. id="p-804" id="p-804" id="p-804" id="p-804" id="p-804" id="p-804" id="p-804" id="p-804" id="p-804" id="p-804"
[804] In T-cell epitope #5 (see Table 7), the arginin nativelye located at position 179 in the matur Ae Subunit of Shiga-like toxin 1 (SEQ ID NO: I) and Shiga toxin (SEQ ID NO:2) was mutated to alani ne(R179A), the threoni natine vely locat ated position 180 in the matur Ae Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutated to glycine (T180G), the threonine natively located at position 181 in the matur Ae Subunit of Shiga-like toxi nI (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) was mutat edto isoleucine (T181I), and the aspartate native locatedly at position 183 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO: I) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (D183A) and to glycine (D183G).WO 2016/196344 PCT/US2016/034778 312 805. 805. 805. id="p-805" id="p-805" id="p-805" id="p-805" id="p-805" id="p-805" id="p-805" id="p-805" id="p-805" id="p-805"
[805] In T-cell epitope #6 (see Table 7), the cysteine natively located at position 242 in tiie matur Ae Subuni tof Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO: 2) was mutated to serine (C242S), the arginine natively located at position 248 in the matur Ae Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (R248A), and the arginine natively located at position 251 m the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to alani ne(R251A). 806. 806. 806. id="p-806" id="p-806" id="p-806" id="p-806" id="p-806" id="p-806" id="p-806" id="p-806" id="p-806" id="p-806"
[806] Furthermor truncae, ting the carboxy-terminus of SET-1A to amino acids 1--- 251 of SEQ ID NO: 1 removed the las twot epitope regions (Tabl e7, #9 and #10), the las CD4+t T-cell epitope (Tabl e6, #7), and the highest scoring discontinuous, B- cell epitope predicte byd ElliPro (289-293). In addition, the truncation at position 251 disrupts T-cell epitope #6 and epitope region #8 (Tabl e7). 807. 807. 807. id="p-807" id="p-807" id="p-807" id="p-807" id="p-807" id="p-807" id="p-807" id="p-807" id="p-807" id="p-807"
[807] An exempla ry,de-immunize Shigad, toxin effector polypeptide of this Example is SLT-lA-combo22 (SEQ ID NO:28), which has seven, amino aci d residue substitutions relat iveto the wild-type Shiga-like toxin 1 A Subunit (SEQ ID NO: 1) and all of thes substitutie onswer predicte toed disrup endogenoust epitopes .
In accord wit hthe labels in Table 8, the Shiga toxin, effector polypept ideSLT-1A- 00mb022 compris aes substitution disrupting epitope region 4; a substitution disrupting epitope region 5; a substitution disrupting epitope region 6; a substitution disrupting epitope region 7; substituti onsdisrupting epitope region 8; epitope regio ns8, 9, and 10 disrupted by truncat ion;and substituti onsdisrupti theng furin- cleava sitege at the carboxy -terminus of the Al fragment-derived region. In addition, SLT-lA-combo22 compris aes substitution disrupting T-cell epitope #2, a substitution disrupting T-cell epitope #4, substituti onsdisrupting epitope #6, and T- cell epitop es#6 and #7 disrupted by truncation.
E. Testing for Reductions in Antigenicities of Exemplary7, Shiga Toxin Effector Polypeptides SLT-lA-combo(n) Using ELISA and Western Blot Assays 808. 808. 808. id="p-808" id="p-808" id="p-808" id="p-808" id="p-808" id="p-808" id="p-808" id="p-808" id="p-808" id="p-808"
[808] Routine methods may be used to evaluate the relat iveantigenic itiof esShiga toxin effector polypepti desin the context of cell-targetin moleculesg (see e.g. WO 2015/113005; WO 2015/113007). The antigenic itiof exees mplar celly, -targeting molecules comprisin cerg tain, combination de-i, mmunized, protease-clea vage resistant, Shiga toxin effector polypepti desSLT-lA-combo( wasn) assess edby Western blots and ELIS As using both polyclonal and monoclo antibonal dies that WO 2016/196344 PCT/US2016/034778 313 bind with high-affini toty the wild-type Shiga-like toxin Al fragment (SLT-1A1).
The cell-targete molecd ule SLT-lA-FR::scFv- (SEQl ID NO:34) was used as a refere ncemolecule. 809. 809. 809. id="p-809" id="p-809" id="p-809" id="p-809" id="p-809" id="p-809" id="p-809" id="p-809" id="p-809" id="p-809"
[809] The Western analys usedes here indetermined relative antigenicit undery' denaturing conditions wherea thes ELISA analyses used herei measn ured relati ve antigenicit undery native protei foldingn conditions. 810. 810. 810. id="p-810" id="p-810" id="p-810" id="p-810" id="p-810" id="p-810" id="p-810" id="p-810" id="p-810" id="p-810"
[810] For Western analyses exempl, ary', cell-target molecing ules comprising the Shiga toxin effector polypeptides SLT-lA-combo SLT-lA-c7, ombolO, or SLT-1A- combo 14 were tested and compared to the results for the reference molecule SLT- lA-FR::scFv-l . Samples of the aforementi onedmolecule wers eloade ind equal amounts to replica 4-20%te, SDS polyacrylamide gels (Lonza, Basel, CH) and electrophorese under ddenaturing conditions. The result inggels were either analyzed by Coomassie staining or transfer tored polyvinyl difluoride (PVDF) membranes using the iBIot® (Life Technologi Carles, sbad, CA, U.S.) system according to manufactur’s instruer ctions Ure. result ingmembranes wer probede under standar condid tions using the followi antibodieng mouses: monoclo nala-Stx (mAbl )(BEINR-867 BEI Resourc es,Manass as,VA, U.S.; cros reas ctive with the Shiga-like toxin 1 A Subunit), rabbit polyclonal antibody a-SLT-lA (pAbl) (Harl an Laboratorie Inc.s, Indianapolis, IN, U.S., custom antibody producti raion se againstd the yvild-type SLT-LI 1, and rabbit polyclonal antibody a-SLT-lA (pAb2) (Genscript, Piscataway, NJ, U.S., custom antibody productio whichn) was raised against peptides from the wild-type Shiga toxin Al fragme nt:RGIDPEEGRFNN and HGQDSVRVGR. The peptide sequence RGIDPEEGRFNN is located at amino acid 55-6s 6 and the peptide sequence HGQDSVRVGR is located at 214-223 in SLT-1A and StxA. Membrane bound antibodies wer edetecte usingd standard conditions and, when appropriat usinge, horseradis peroxidaseh (HRP) -conjugated secondary antibodies (goat anti-rabbit-H orRP goat anti-mouse-HR ThermoP, Scientific Rockf, ord, IL, U.S.). Figur e13 shows Western blots with the lane ofs the gels and/or membranes numbered and the figur legee nd indicating by the same respectiv numbee ring which Shiga toxin effector polypeptide regions were present in the cell-target moleingcule load edinto each lane. Coomassie stained lane ares shown as sample loading controls. 811. 811. 811. id="p-811" id="p-811" id="p-811" id="p-811" id="p-811" id="p-811" id="p-811" id="p-811" id="p-811" id="p-811"
[811] Figure 13 shows tliat, under denaturing conditions, exempla cellry' -targetin g molecules SLT-lA-combo7::scFv-l, SLT-lA-combolO::scFv־l, and SLT-1A- WO 2016/196344 PCT/US2016/034778 314 combo 14: :scFv-1 exhibit decreas antied genic itias comparedes to SLT-lA-FR::scFv- 1 in this assa y.These results demonstr thatate SLT-lA-combo SLT-lA-c7, ombolO, and SLT-1 A-combol4 have decreased antigenic itieas compars edto SLT-1A-FR, or by inference to SLT-1 A-WT, when linked by the same fashion to the same targeti ng domain (scFv-1) using the same linker Addit. iona lly,the results shown in Figur e13 suggest that SLT-1 A-combo7 has reduced relati antigeve nicit as compay red to SLT- lA-combolO and SLT-1 A-combo 14 in this assa undery the conditions teste . d 812. 812. 812. id="p-812" id="p-812" id="p-812" id="p-812" id="p-812" id="p-812" id="p-812" id="p-812" id="p-812" id="p-812"
[812] A standar ELISAd was used to measure the ability of a-SLT-1 A mAbl to recognize vario usde-immunize Shigad, toxin effector polypeptide eas,ch with multiple epitope regio nsdisrupted, in the context of a cell-targetin molecg ule. The ability of each cell-target moleingcule teste tod bind the target biomolecul of eits scFv binding region was utilize ind the assa y.The wells of Nunc MaxiSorp® plate s m phosphate buffered saline (IX PBS) (Hyclone Bran d,Fisher Scientific Walth, am, MA, U.S.) wer ecoat edwith recombinant, human targe biomolt ecul ofe the binding regio (scFvn -1). The plate wers eincubated overnight at 4°C. The wells were washe withd IX PBS 0.05% Tween-20 (PBS-T), and non-specifi bindinc wasg blocke byd incubati ngthe wells with 3% milk in PBS-T for one hour at room temperat ure.Exemplar cell-y target molecing ules wer eadded to the wells where, certain well receis ved only one cell-target moleingcule comprisin onlyg one, combination de-i, mmunize Shigad, toxin effector polypeptide SLT-lA-combo(n): SLT-1 A-combo7: :scFv1,- SLT-1 A-combo 10::scFv-1, or SLT-1 A-combo 14::scFv-1.
In addition, the cell-targeted molecule SLT-1A-FR::scFv-1 was added to certa in wells as a refere ncemolecule. Ail cell-targeti molecng ules wer eadded to the wells at a concentrat deteionrmine to dbe above tli emaximu mbindin g(Bmax) determine d previous witly han ELISA using Protein L conjugated to HRP to detec SLT-1A-t FR::scFv-l, thus allowing for 100% of the availa bletarge biomolt ecule to bes bound by the cell-target moleingcule sample, whic his in excess. The plate weres incubate d at room temperature for one hour to allow7 for cell-target moleculeing to bind to targe biomolecult undere non-denaturing conditions. The wells wer ewashed with PBS-T and then incubated with anti-SxtA mouse monoclo antibodynal conjugated to HRP (anti-SLT-lA mAbl-HRP) or rabbit polyclona antil body a-SLT-1 A conjugated to HRP (anti-SLT-1 A pAb2-HRP) or protein L-HRP (which binds to scFv-1 and was used as a loadin control)g for 1 hour at room temperat ure.The wells were washed in PBS-T then incubated with Pierc TMBe Ultra (Thermo Scientifi Inc.,cRockford, IL, U.S.). The reactions were stoppe witd h250 mM hydrochloric acid (HC1). HRP activi wasty detected in the wel lsby adding a chromogeni HRPc substrat ande then detect inglight emission, result ingfrom chemiluminescenc usinge, a plate reading devic measurie ngabsorbance (Abs) of light set to the wavelength of 450 nanomete (nm).rs 813. 813. 813. id="p-813" id="p-813" id="p-813" id="p-813" id="p-813" id="p-813" id="p-813" id="p-813" id="p-813" id="p-813"
[813] The measured absorbance values were correcte for dbackground by subtract theing absorban valuesce for coated, blocked wel lsincubated wit honly PBS instea ofd any cell-targeting molecul same ple. To normal theize signals from the three different detec, tio antin bodie thes, signa lsfrom SLT-1 A-FR::scFvl was set at 100%, and the relat ivesignals as a percentage of this contr wereol determined for each cell-target molingecule sample test edby the calculation (Abs signal of the sampl /e Average Abs signal of the control) x 100. The ELISA resul arets shown in Figure 14. 814. 814. 814. id="p-814" id="p-814" id="p-814" id="p-814" id="p-814" id="p-814" id="p-814" id="p-814" id="p-814" id="p-814"
[814] Figure 14 shows that under, native conditions, the exemplary cell-targeti ng molecules SLT-lA-combo7::scFv-l (SEQ ID NO:44), SLT-1 A-combol0::scFv-l (SEQ ID NO:47), or SLT-lA-combol4::sc Fv-l(SEQ ID NO:50) exhibit decreased antigenici asti comparedes to SLT-lA-FR::scFv-l (SEQ ID NO:34). These resul ts demonstrat that eSLT-1 A-combo7, SLT-1A-combo10 and, SLT-1 A-combo 14 exhibit decreased antigenicit asies compared to SLT-1A-FR (SEQ ID NO:5), or by inference or SLT-1 Al-WT (SEQ ID NO:4), when linked by the same fashion to the same target domaiing (scFv-n 1) using the same linker. Additionall the y,resul ts shown in Figure 14 suggest that SLT-1 A-combo7 and SLT-1 A-combo 14 have reduced relati antigeve nicit asies compared to SLT-1 A-combo 10 under the native conditio ofns this ELISA assay.
F. Testing the CD4+ T-Cell De-Immunizatio ofn Exemplary, Shig aToxin Effect or Polypepti desSLT-lA-combo(n) 815. 815. 815. id="p-815" id="p-815" id="p-815" id="p-815" id="p-815" id="p-815" id="p-815" id="p-815" id="p-815" id="p-815"
[815] Disruptions in predicte CD4+d T-cel epitopel regions are test edfor reductions in CD4+ T-cell immunogenicity using assa ysof human CD4+ T-cel l proliferati in theon presence of exogenousl admiy nistered polypepti desand assays of human CD4+ dendriti T-cellc stimulat inion the presenc ofe human monocytes treat wited hadministered polypeptides. 816. 816. 816. id="p-816" id="p-816" id="p-816" id="p-816" id="p-816" id="p-816" id="p-816" id="p-816" id="p-816" id="p-816"
[816] T-cel prolil ferat assaion ysknown to the skilled worker are used to test the effectivene of CD4+ss T-cell epitope de-immunizati ofon Shiga toxin effector -315-WO 2016/196344 PCT/US2016/034778 316 polypepti desSLT-lA-combo(n) The. T-cell proliferation assay of this Example involves the labeling of CD4+ T-cells and then measurin chang ges in prolifera tion using flow cytometric method ins respons toe the administrat ofion differe nt peptides derived from either a Shiga, toxin effector polypept idecombo(n) or a refere ncemolecul suche, as, e.g., a wild-type Shiga toxin Al fragment, SLT-1A-FR, and/o ar related cell-targetin molegcule comprisin theg aforementioned. 817. 817. 817. id="p-817" id="p-817" id="p-817" id="p-817" id="p-817" id="p-817" id="p-817" id="p-817" id="p-817" id="p-817"
[817] A series of overlappi peptidesng derived from the chosen molecule are synthesize andd tested in the CFSE CD4+ T cei lprolifera asstionay (Prolmmune Inc., Sarasota, FL, U.S). Human CD8+ T-cell depleted, peripher bloodal mononuclear cells (PBMCs) labeled wit hCFSE are cultured with 5 pM of each peptide of intere forst seve ndays in six replic atewells. Each assay plat include esa set of untrea tedcontrol wells. Hie assay als oincorpor aterefsere nceantigen contro comprls, isin synthetg peptidesic for known MHC class II antigens or agretopes. 818. 818. 818. id="p-818" id="p-818" id="p-818" id="p-818" id="p-818" id="p-818" id="p-818" id="p-818" id="p-818" id="p-818"
[818] The CD8+ T-cell depleted, PBMCs that proliferate in respons toe an administer peptideed will show' a reduction in CFSE fluorescenc intensitye as measure directd byly flow cytometry. For a naive T-cell analys theis, Percenta ge Stimulation above background is determine ford eac hstimulated sample, through comparison with results from an unstimulate samd ple, such as by ranking wit h rega rdto fluoresce signalnt as, negative dim,, or high. Counts for the CD4+ CFSE T-cell dim population in each sample are expresse asd a proportion of the total CD4+ T-cell population The. replic atevalues are used to calcul atePercentage Stimulation above Background (proportio of nCD4+ T-cell CFSE dim cells with antigen stimulat ion,minus proporti ofon CD4+ T-cell CFSE dim cells without antigen stimulati on).The mean and standa errrd or of the mean are calculat fredom the replic atevalue s.A result is considere ‘"positived " if the Percentage Stimulation above background is great thaner 0.5% and also great thaner twice the standar errd or above background. To allow for comparison of peptide as, Response Index is calculate Thisd. index is based on multiplying the magnitude of response (Percenta Stimulage tion above background) for eac hpeptide by the number of responding donors (Percentage Antigenici forty) eac hpeptide.WO 2016/196344 PCT/US2016/034778 317 G. Determin ingthe Relative, CD4+ T-Cell Immunogeniciti of esExempla ryShiga Toxin Effector Polypeptide SLT-lA-cs ombo(n) 819. 819. 819. id="p-819" id="p-819" id="p-819" id="p-819" id="p-819" id="p-819" id="p-819" id="p-819" id="p-819" id="p-819"
[819] The relat iveCD4+ T-cel immunl ogenic ofity molecules of the invention is determined using the following dendritic cel (DC)l T-cell proliferat assionay. Uris DC T-cell assay measur esCD4+ T-cell responses to exogenously administere d polypepti desor protein lires, DC T-cell assay is performed using Prolmmune’s DC-T assa servicey to determi thene relat ivelevels of CD4 + T-cell drive n immunogenicit betweeny proteins and cell-targetin molecg ules of the present invention as compar toed reference molecules lire, DC T-cell assay of this Example involves testing human dendritic cells for antigen presentati ofon pepti des derived from the administered polypepti protein,de, or cell-targetin molegcule samples. 820. 820. 820. id="p-820" id="p-820" id="p-820" id="p-820" id="p-820" id="p-820" id="p-820" id="p-820" id="p-820" id="p-820"
[820] Briefly, healthy human donor tissues are used to isola typete dsamples base d on high-resolution MHC class II tissue-typing. A coho ofrt 20, 40 or 50 donors is used. First monocyte, obtains edfrom human donor PBMCs are cultured in a defined medium to generat immate ure dendritic cell s.Then, the immatur dendritice cel lsare stimulated with a well-defined contr antigenol and induced into a more mature phenotype by further culture in a defined medium. Next, CD8+ T-cell depleted donor PBMCs from the same human donor sample are labeled with CFSE.
The CFSE-labeled, CD8+ T-cell depleted PBMCs are then cultured with tire antigen-primed, dendritic cells for seve ndays to allow for CD4+ dendritic cell stimulat ion,afte whichr eight replicat fores each sample are tested. As negative contro eachls, dendritic cell culture series also includes a set of untrea teddendritic cell s.For a positive control the ,assay incorporat twoes well-define referenced antige ns,eac hcomprisin a gfull-lengt proteih n. 821. 821. 821. id="p-821" id="p-821" id="p-821" id="p-821" id="p-821" id="p-821" id="p-821" id="p-821" id="p-821" id="p-821"
[821] To evaluate dendritic cell base dimmunogenici thety, frequency of donor cell responses is analyzed across the study cohor Positivet. response in sthe assay are considere indicad tive of a potenti inal vivo CD4+ T-cell response. A positive response, measure asd a percenta ofge stimulat aboveion background, is defined as percentage greats thaner 0.5 perce nt(%) in two or more independent donor samples .
Tire strengt of hpositive donor cell responses is determined by taking the mean percentage stimulat aboveion background obtain edacross accepted donors for each sample. A Response Index is calcula byted multiplying the value of the strength of respons bye the frequency' of the donors responding to determi levelsne of CD4+ T- cell immunogenicit for yeach sample. In addition, a Response index, representing WO 2016/196344 PCT/US2016/034778 318 tiie relat iveCD4+ T-ceil immunogenicit is detey rmine by dcomparing the results from two samples, one involving a Shiga Toxin Effector Polypeptide SLT-1A-s combo(n) and a second variant which is a relate moled cule that lacks one or more predicted disruptions of a. CD4+ T-cell epitope and/or epitope region as a referenc e molecule.
H, Testing For Reductions in Immunogeniciti ofes Exempla ry,Cell-Targeting Molecul Comprisinges Shiga Toxin Effector Polypeptide SLT-lA-cs ombo( n) 822. 822. 822. id="p-822" id="p-822" id="p-822" id="p-822" id="p-822" id="p-822" id="p-822" id="p-822" id="p-822" id="p-822"
[822] Mice wer eused to investigate the immunogeni potentic ofal certa in exempla moleculery' ofs the prese ntinvention . The relat iveimmunogenicitie of s exemplar cell-y target molecing ules were determined using an assa fory in vivo antibody response to sthe cell-target molecing ules aft errepeat, parente ral administrati overons periods of many weeks (see e.g. WO 2015/113005). An in- solution ELISA was used to determ inethe relat iveamount of serum murine antibodies that wer especif icto differ entcell-target molecules.ing This immunogenicity assay involves the use of mice which are indicative of the relati ve immunogenicitie of molecs ules in mammals generally. 823. 823. 823. id="p-823" id="p-823" id="p-823" id="p-823" id="p-823" id="p-823" id="p-823" id="p-823" id="p-823" id="p-823"
[823] This assay was used to determi thene relati immve unogenicit of exemplay ry' cell-target moleculeing comprs isin SLT-lAg ־combo(n)::scFv-(n) as compared to the les sde-immunize cell-d, targe moletedcule SLT-lA-FR::scFv(n) or to the reference molecule scFv-3::SLT-lA-W (SEQT ID NO:33). The refere ncemolecule scFv- 3::SLT-1A-WT was construct in edthe reverse, amino-carboxy fusion orientat ofion tire exemplar ceyll-targeti moleculeng ofs the prese ntinvention teste ind the relat ive immunogenicit assyay, and the Shiga toxin effecto polypeptider component of scFv- 3:: SET-1A-WT consisted of a wild-type Shiga toxin A1 fragment (SEQ ID NO:4), which represe ntsan "un-de-immunized" Shiga toxi effen ctor polypeptide. 824. 824. 824. id="p-824" id="p-824" id="p-824" id="p-824" id="p-824" id="p-824" id="p-824" id="p-824" id="p-824" id="p-824"
[824] Four differ entmouse studie weres conducte whered BALB/c or C57BL/6 mice wer erandomly assigned to treatment groups consist ingof six mice per group and where the mice in differ enttreatment groups wer eadminister differented cell- targeti moleculesng First. serum, samples were collect fromed each mouse prior to exposure to a cell-target moleingcule Next,. each mouse in a treatment group was administer 0.25ed milligram of the sample molecule per kilogr amof body weight (mg/kg) per dose of the sample cell-targeti molengcule by intra-peritoneal injecti on three time as week for two weeks. After a week without administration of any WO 2016/196344 PCT/US2016/034778 319 samples, intra-peritoneal injections of 0.25 mg/kg per dose of the sample cell- targeting molecule were administered three time as week for an additional two weeks, result ingin a total of 12 doses of cell-target moleingcule over a five-we ek interval For. all studie thes, administrate moleculesd SET-1 A-combo 1: :scFv- 1(SEQ IDNO:43), SLT-1 A-combo7 scFv-1:: (SEQ IDNO:44), SLT-1 A-combo 10::scFv- 1(SEQ ID NO:47), SLT-1 A-combo 10::scFv-2 (SEQ ID NO:61), SLT-1A- combo 12: :scFv-1 (SEQ ID NO:49), SLT-1 A-combo 15::scFv-1 (SEQ ID NO:51), SLT-lA-combol6::sc Fv-l(SEQ ID NO:52), SLT-1 A-combo 19::scFv-1 (SEQ ID NO:55), SLT-lA-combo22::scFv-2(SEQ ID NO:63), and the refere ncemolecule s SET-1A-F.R:: scFv-1 (SEQ ID NO:34) and SLT-lA-FR::sc.Fv-2 (SEQ ID NO:35) wer ewell-toler resultated, ingin no or only minimal effects on body weight and no clinical sign s.During and aft erthe five-w'eek administration interval, sera were collect fromed all the mice to observe antibodie tars geting the administer cell-ed targeting molecules using in-soluti ELISAson . The mouse studie weres done at Charles River Laborator inies Piedmont, NC, U.S. Tire results of thes studiese are shown in Table 13-16s and Figures 15-16. 825. 825. 825. id="p-825" id="p-825" id="p-825" id="p-825" id="p-825" id="p-825" id="p-825" id="p-825" id="p-825" id="p-825"
[825] The in-solution ELISAs to dete ctantibodies recognizi theng administer ed cell-targetin molecg ules were specif icto the bindin gregio scFv-(n n) of the ceil- targeting molecule being test edand performed as follows For. each cell-targeting molecule and its respectiv mousee -treatment group, a different ELISA assa wasy performed but using the same general in-solution ELISA assay setup .For eac hcell - targeting molecule and its respectiv mousee -treatm group,ent the in-solution ELISA assay setup involved only the appropriate targe biomolecult ofe the scFv-(n) of the cell-targetin molegcule of that group. For all in-solution ELISA assays, the ELISA plate wells wer coatee withd a targe biomolecult of ethe cell-targe bindingting, regio sc-Fn v-(n ).The same cell-target moleingcule used for injectio inns a mouse- treatme groupnt was incubated overnight at 4°C in solution with the serum collected from a singl mousee from, that group, and then any complexes formed (e.g. complexes comprisin theg cell-targeti molengcule and antibodies prese ntin the serum) wer capturede using the coated ELISA plat wells.e Captured, immune complexes comprising murine, immunoglobulin G molecules (IgGs) wer edetecte d using an anti-mouse IgG, secondary־ antibody conjugated to horsera dishperoxidas e.
HRP activ itywas detecte in dthe wells by adding a chromogeni HRPc substrat ande then detectin lightg emission as a result of chemiluminscenc Thee. reaction was WO 2016/196344 PCT/US2016/034778 320 stopped by the addition of HC1, and HRP activity or "ELISA signal" was measured as at 450 nM using a plat reae der. ELISA signal values were calculated as Absorbance value (Abss 450 nM) after subtracti theng background signa asl measured from "no serum" negat ivecontrol wells. Serum was diluted to allow for Absorbance value readings below the level of saturation for the assa y,and the dilution rat iowas tire same for all mice in all treatment groups measured on a give n day. For the genera setl up of thes in-se olution ELISA assays, larger ELISA signa l value indicas thete presence of more murine IgG antibodie recs ognizi theng administer ceedll-targeti molecng ule or in othe wordsr great immunogenicity.er 826. 826. 826. id="p-826" id="p-826" id="p-826" id="p-826" id="p-826" id="p-826" id="p-826" id="p-826" id="p-826" id="p-826"
[826] Based on these in-solution ELISA assays, none of the mice in any treatme nt group of the four studie wers eobserve tod have pre-forme serumd antibodies recognizi theng cell-target molecing ules tested prior to exposure via injection. Thus, any post-administration detectio of nant1-"cell-targeti molengcule" IgG antibodies in the sera of, the mice of thes studiee usings the in-soluti ELISAon assay represents de novo, antibody producti inducon ed after the administrat ofion a cell-targetin g molecule. 827. 827. 827. id="p-827" id="p-827" id="p-827" id="p-827" id="p-827" id="p-827" id="p-827" id="p-827" id="p-827" id="p-827"
[827] Murine IgG antibody response to sthe administer cell-ed target molecing ules wer emeasured in the four studie ats different time-points using the appropriate in- solution ELISA assays, and the results are reported in Table 13-16s and Figures 15- 16. At each time-point serum, samples wer ediluted such that tire ELISA signal value remas ined within the dynami rangec of the assa y.For a single time-poin int an individual study all, serum samples wer edilute identicd ally. The average ELISA Absorbance values for eac hmouse-treatm groupent at individual serum collection time-points ("avera ELISAge signal") wer ecalcula ted.In conducti thesng rele ati ve immunogenicit studiey thes, same cell-targetin molegcule injected into the mice of a particula treatmentr group was used in the ELISA assay to capture ser antibodiesa from ser acollect onlyed from the mice of tliat same group. In other words, anti- "cell-target moleingcule" IgG antibodie preses ntin the ser afrom mice administer ed SLT-1 A-FR::scFv-l (the SLT-1 A-FR::scFv-l reference group) were captured and detecte in dan ELISA assa designey specd ifica andlly only with SLT-lA-FR;:scFv- 1, and, similar anti-ly, "cell-target moleingcule" IgG antibodies prese ntin the sera from mice administer SLT-lA-ed combo7::scFv-l (e.g. the SLT-1 A-combo7::scFv-l group) wer capturede and detecte usingd an in-solution ELISA assa desiy gned specifica andlly only with SLT-lA-combo7::scFv-l.WO 2016/196344 PCT/US2016/034778 321 828. 828. 828. id="p-828" id="p-828" id="p-828" id="p-828" id="p-828" id="p-828" id="p-828" id="p-828" id="p-828" id="p-828"
[828] The average ELISA signal value fors each mouse-treatm groupent at individual serum collection time-points were calcula ted,and then the relati ve immunogenicit wasy calculat fored eac hgroup at each time-point relat iveto the average ELISA Absorbance value at the same respectiv time e-point for the refere nce group treated wit hthe reference cell-target moleculeing as describe above.d The relati immve unogenici ofty each, tested, exemplar cell-y target moleingcule (SET- lA-combo(n)::scFv-( inn)) a given study at a certain time-point as compared to the immunogenicit of ay refere ncemolecule (e.g., SLT-lA־FR::scFv-(n) or scFv- 3::SLT-1A-WT) was calculat usinged the formula (ELISA: signa ofl cell-targeting molecule - average ELISA signa ofl "no serum." contr ol)/ (average ELISA signa ofl refere ncemolecule - average ELISA signa ofl "no serum" contr ol)x 100. To creat e Figures 15-16, the percentag of thee refere ncemolecule ELISA signa forl each mouse-treatment group was graphed on the Y-axis, and the day of serum collect ion was graph edon the X-axis .To measure mammalia IgGn responses to each cell- targeting molecule, the average ELISA signal ("avg signal") and the perce ntof the avg signa ofl the reference molecule SLT-lA-FR::scFv-(n) ("perce ntof ref") were calculated for each mouse-treatme groupnt (Tables 13-16). 829. 829. 829. id="p-829" id="p-829" id="p-829" id="p-829" id="p-829" id="p-829" id="p-829" id="p-829" id="p-829" id="p-829"
[829] The results of the relat iveimmunogenicit assay ysfrom the first mouse study are presented in Figure 15-panel A and Table 13. The term "N/A" was used to indica "tenot applicable" becaus calce ulati atons Day 1 involved pre-treat ment serum, with ELISA signa valul esof zero.
Table 13. Relative Immunogeniciti ofes the Exemplary', Cell-Targeting Molecule SLT-1 A-combo7:;scFv-l Compared to SLT-lA-FR::scFv-l Reference SLT-lA-combo7: :scFv-1 SLT-1A-FR: :scFv-1 Day of serum avg ELISA percenta ofge avg ELISA percentag of e collectionsignal signal reference reference 0 N/A 0 100 1 0.02 3.90% 0.49 100 0.10 11.20% 0.87 100 22 0.20 27.30% 0.73 100 29 36 0.33 35.60% 0.93 100 0.20 24.60% 0.80 100 43 0.19 26.30% 0.73 100 50WO 2016/196344 PCT/US2016/034778 322 830. 830. 830. id="p-830" id="p-830" id="p-830" id="p-830" id="p-830" id="p-830" id="p-830" id="p-830" id="p-830" id="p-830"
[830] The results of the first, relat iveimmunogenicity, mouse study wer thate the exempla ry,cell-targeti molengcule SLT-lA-combo7::scFv- exhibitedl reduced immunogenicity as compared to the reference cel, l-target moleedcule SLT-1A- FR::scFv-l at all time-point Thes. average ELISA Absorbance value fors the SET- lA-combo7::scFv-l treatme groupnt were lower than for the SLT-lA-FR::scFv-l treatme groupnt at all time-points afte Dayr 1 (sera was collected pre-treatme on nt Day 1). For the cell-targeti moleculeng testes anti-d, ‘،cell-targe molecting ule" IgG response weres first observe ond Day 15 (3 days aft erthe administra oftion the 6th dose), and, then, murine IgG response weres observe atd all subsequent time-point: s Day 22 (10 days afte ther administration of the 6th dose, prior to administration of the 7m dose), Day 29 (3 days after the administrat ofion the 9th dose prior to administra oftion the 10th dose) Day, 36 (3 days afte tirer administration of the las t dose), Day 43 (10 days after the administration of the 11th dose), and Day 50 (17 days after the administrat ofion the las dose)t (Figur e15-panel A; Table 13). The data in Table 13 showed that the cell-targetin molegcule SLT-lA-combo7::scFv-l comprising the de-immunize furd, in-cleavage resista Shigant, toxin effector polypept idecomb7o had reduce immd unogenicit as comparedy to the cell-targetin g molecule SLT-lA-FR::scFv-l comprising a wild-type Shiga toxin Al fragment.
These results suggest that molecules comprising only Shiga toxin effector polypepti desthat consi stof the combination de-im, munize protead, clease vage- resistan Shigat, toxin effector polypeptide scaffol combo?d exhibit reduced immunogenicit compay red to molecules comprisin a gShiga toxin effector polypeptide comprising (1) a wild-type, Shiga-like toxin Al fragment or (2) the furm-cleavage resistant, Shiga toxin effector polypeptide SLT-1A-FR. 831. 831. 831. id="p-831" id="p-831" id="p-831" id="p-831" id="p-831" id="p-831" id="p-831" id="p-831" id="p-831" id="p-831"
[831] The results of the relat iveimmunogenicit assay ysfrom the second mouse study are shown in Figur e15-panel B and Table 14.WO 2016/196344 PCT/US2016/034778 323 Table 14. Relative Immunogenicities of Exemplary, Cell-Target Moleing cule s Comprising De-immunize d,Protease-Cleavage Resista nt,Shiga Toxin Effector Polypeptides Reference SLT-1A- SLT-1A- SLT-1A- SLT-1 A- com bo 19:: scFv-1 combolO::scFv-lcombol6::scFv-l FR::scFv-l Day of serum avg percent of avg perce ntof avg perce ntof avg collectionsignal ref signal ref signal ref signal 1 0 N/A 0 N/A 0 N/A 0 0.26 13.5 0.36 19.2 1.21 63.7 1.9 22 0.44 24.3 0.44 24.4 1.29 71.2 1.81 29 0.27 19.5 0.71 51.5 1.21 87.2 1.38 1.10 71.2 36 0.59 38.4 1.46 94.6 1.55 1.38 71.5 40 0.83 43.3 2.06 106.8 1.93 832. 832. 832. id="p-832" id="p-832" id="p-832" id="p-832" id="p-832" id="p-832" id="p-832" id="p-832" id="p-832" id="p-832"
[832] Ilie results of the second relat, iveimmunogenici mousety, study wer ethat the exemplary, cell-targetin moleculesg teste (SLT-d lA-combolO::scFv-l, SLT-1A- combol6::scFv-l, and SLT-1A-combo19:scFv-1 exhibite) reducedd immunogenici asties compared to the reference cell-targete moledcule SLT-1A- FR::scFv-l at all time-points up to Day 36 (Figur e15-panel B; Table 14). For the cell-targetin molecg ules tested, anti-،‘cell-targeting molecule־’ IgG response weres first observe ond Day 15 (3 days aft erthe administration of the 6th dose), and, then, murine IgG responses were observed at all subseque nttime-point Days: 22 (10 days after the administration of the 6th dose, prior to administrat ofion the 7th dose) Day, 29 (3 days after the administrat ofion the 9th dose prior to administrat ofion the 10th dose), Day 36 (3 days after the administra oftion the las dose),t and Day 40 (7 days after the administration of the last dose (Figur) e15-panel B; Table 14). At both Day and Day 22, the mice in the group administer theed refere ncecell-targete d molecule SLT-lA-FR::scFv-l exhibite highed magnitudesr of total IgG antibody responses as shown by the ELISA signal than the mice m the groups administere d exempla cell-ry' target molecing ules comprising the de-immunize protead, se-clea vage resistant, Shiga toxin effector scaffolds (SLT-1 A-combolO, SLT-1 A-combol6, and SLT-lA-combol9) (Figur e15; Table 14). At Days 29, 36, and 40, the percentages of the average refere nceELISA signal of the average ELISA signa valuel fors the groups administere SLT-lA-cd ombol6::sc Fv-land SLT-lA-combol9::sc Fv-lwere highe thanr for the group administer SLT-ed 1A-combo 1:: scFv-1. At all time-points, tiie percenta ofge the average reference ELISA signa ofl the average ELISA signalWO 2016/196344 PCT/US2016/034778 324 value fors the SLT-lA-combol9::scFv-l group was higher than the percentage of tiie average ELISA signal value fors the group administer SLT-1A-ed combo 10:: scFv-1 or SLT-1 A-combo 16:: scFv-1. 833. 833. 833. id="p-833" id="p-833" id="p-833" id="p-833" id="p-833" id="p-833" id="p-833" id="p-833" id="p-833" id="p-833"
[833] The results of the relat iveimmunogenicit assay ysfrom the third mouse study are displayed in Table 15 and Figure 15-panel C.
Table 15. Relative Immunogenicities of the Exempla ry,Cell-Target Moleing cules . Each Other Compared to Reference Molecules anc reference 1 refere nce2 SLT-lA-combolO: :scFv-2 scFv-3: SLT-1A-FR SLT-1A-WT ::scFv-2 Day of perce ntof perce ntof serum avg ELISA ref 2 SLT-1A- avg ELISA avg ELISA ref 1 scFv- collectionsignal 3::SLT-1A-WT FR:: scFv-2 signal signal 0 N/A N/A 0 0 1 0.11 7.4% 4.9% 1.49 2.25 0.27 12.3% 9.6% 2.20 2.81 22 1.28 40.6% 35.2% 3.15 3.64 29 SLT-lA-combo22: :scFv-2 percent of Day of perce ntof reference 1 referenc 2 e serum avg ELISA ref 1 scFv- ref 2 SLT-1A- avg ELISA, avg ELISA collectionsignal 3::SLT-1A FR:: scFv-2 signal signal 0 N/A N/A 0 0 1 0.60 40.3% 26.7% 1.49 2.25 1.13 51.4% 40.2% 2.20 2.81 22 2. !5 86.7% 75.0% 3.15 3.64 29 refere nce3 SLT-lA-combolO :sc:Fv-2 SLT-lA-combo22: : scFv-2 Day of perce ntof serum avg ELISA avg ELISA ref 3 SLT-1A- collectionsignal combo22:: scFv-2 signal 0 N/A 0 I 0.11 18.3% 0.60 0.27 23.9% 1.13 22 1.28 46.9% 2.73 29 834. 834. 834. id="p-834" id="p-834" id="p-834" id="p-834" id="p-834" id="p-834" id="p-834" id="p-834" id="p-834" id="p-834"
[834] The average ELISA Absorbance value fors the SLT-1A-combo 10: :scFv-2 and SLT-1 A-combo22:: scFv- 2treatment groups wer elower than for the SLT-1 A-WO 2016/196344 PCT/US2016/034778 325 FR::scFv-2 and scFv-3::SLT-lA-W treatmeT groupsnt at ail time-points after Day 1 up to Day 29. Ilie data in Table 15 showed that the cell-targetin molecg ules SLT- lA-combolO::sc־ Fv2and SLT-lA-combo22::scFv-2, eac hcomprisin a gde- immunized, furin-cleava resistge ant, Shiga toxin effector polypeptide, exhibited reduced immunogenicities compared to the ceil-targeting molecule SLT-1A- FR::scFv2 and the wildtype SLT-1 Al fragment in the context of a different scFv (scFv-3) in the rever secarboxy-ami, fusionno orientation. Thes eresults sugges thatt cell-targetin molecg ules whose Shiga toxin effector polypept ideregion consis ofts tire combination de-i, mmunize protead, cleavse age-resis Shigatant, toxin effector polypeptide scaffol combod 10 or combo22 show reduced immunogenicities compared to cell-target molecing ules whose Shiga toxin effector polypeptide region consis ofts (1) a wild-type, Shiga toxin effector polypept ideor (2) the furin-cleavage resistant, Shiga toxin effector polypeptide SLT-1A-FR. 835. 835. 835. id="p-835" id="p-835" id="p-835" id="p-835" id="p-835" id="p-835" id="p-835" id="p-835" id="p-835" id="p-835"
[835] The results of the relat iveimmunogenic assaity ysfrom the four thmouse study are shown in Figur e16 and Table 16.
Table 16. Relative Immunogenicitie of Exempls ary, Cell-Target Moleculing ases Compared to SLT-lA-FR::scv-1I SLT-1A- SLT-1A- SLT-1A- SLT-1A- Reference 1 Day of combolO:comboi l:com bo 15: combol : SLT-1A- serum :scFv-1 :scFv-1 :scFv-1 :scFv-1 FR: :scFv-1 collectio avg avg av8 | perce nt avS percent percent av8 | percent n signal signal | of ref 1 signal of ref] signa! of ref 1 signal 1 of ref l 0 N/A 0 N/A 0 N/A 0 N/A 0 1 0.004 | 1.17 0.136 41,14 0.020 6.09 0.009 2.88 0.329 0.442 61.54 0.023 3.24 0.063 8.71 0.053 7.43 0.719 22 0.937 47.81 0.065 3.33 0.547 27.89 0.215 10.98 1.959 29 36 1.949 94.08 0.551 26.60 1.117 53.90 0.551 26.59 2.072 40 2.247 99.19 0.873 ן 38.55 1.576 69.57 0.832 36.75 2.265 Reference 2 SLT-.1A- SLT-1A- combol combolO::scFv-l ::scFv-1 Day of percent of serum avg ELISA avg ELISA ref 2 SLT-1A- collectionsignal signal combol ::scFv-1 1 0 N/A 0 0.004 2.85% 0.136 0.023 5.27% 0.442 22 0.065 6.97% 0.937 29WO 2016/196344 PCT/US2016/034778 326 0.551 28.27% 1.949 36 0.873 38.87% 2.247 40 836. 836. 836. id="p-836" id="p-836" id="p-836" id="p-836" id="p-836" id="p-836" id="p-836" id="p-836" id="p-836" id="p-836"
[836] The results of the fourt immh unogenicit studyy wer thate the exemplar y, cell-targetin molecg ules tested exhibite reducedd immunogenici asties compared to the refere ncecell-target moleingcule SLT-lA-FR::scFv-l, at leas att earl iertime- point (Figurs e14-panel A; Table 16). Sera, was collected pre-treatme on Daynt 1.
For the cell-targeti molecng ules teste anti-d, "cell-target molecing ule’’ IgG responses wer efirst observe ond Day 15 (3 days afte ther administration of the 6th dose) and,, then, murine IgG responses wer eobserved at all subsequent time-points Day: 22 (10 days after the administrat ofion the 6th dose, prior to administrat ofion the 7th dose), Day 29 (3 days afte ther administration of the 9th dose prior to administration of the ® dose), Day 36 (3 days aft erthe administration of the las dose),t and Day 40 (7 days after the administratio of then las dose)t (Figur e14; Table 16). At both Day , Day 22 and Day 29, the mice in the group administer theed refere ncecell- targeting molecule SLT-lA-FR::scFv-l exhibited highe magnitudesr of total IgG antibody response as sshown by the ELISA signa thanl the mice in the groups administer exemplared cey'll-targeti molecng ules comprisin theg de-immunized, protease-cle avageresistant, Shiga toxin effector scaffolds (SLT-lA-combol, SLT- lA-combolO, SLT-lA-combol2 and, SLT-lA-combo 5)l (Figure 16; Table 16). At all time-point thes, percentage of the avera referencege ELISA signal of the avera ge ELISA signal values for the SLT-1 A-combol::scFv-l treatme groupnt was higher than the percenta ofge the avera ELISAge signa valuel fors the treatment groups administer SLT-lAed -combolO::scF SLT-1v-l, A-combo 12: :scFv-1, and SLT-1A- combo!5: :scFv-1. At Days 36 and 40, the percentag ofes the average reference ELISA signa ofl the average ELISA signa valuesl for the treatmen groupt administere SLT-lA-cd ombol2::sc Fv-lwere higher than for the treatment group administere SLT-ld A-combolO::scFv-l and SLT-lA-combol5::scFv-l. These results suggest that cell-target molecing ules whose Shiga toxin effector polypeptide region consists of the combination, de-immunized, protea clese avage-resis tant, Shiga toxin effector polypeptide scaffold SLT-lA-combol, SLT-1 A-combo 10, SLT- 1 A-combol2, and SLT-1 A-combo 15 show reduced immunogenicit compay red to cell-targetin molecg ules whose Shiga toxin effector polypept ideregion consis oftsWO 2016/196344 PCT/US2016/034778 327 (1) a wild-type, Shiga toxin effector polypeptide or (2) the furin-cleava resistge ant, Shiga toxin effector polypeptide SLT-1A-FR. 837. 837. 837. id="p-837" id="p-837" id="p-837" id="p-837" id="p-837" id="p-837" id="p-837" id="p-837" id="p-837" id="p-837"
[837] Of the cell-targetin moleculeg tess ted in the second third,, and four thmouse studies the, combination de-i, mmunize proteasd, e-cle avageresistant, Shiga toxin effector scaffold SLT-lA-combolO appeared to be the most de-immunized by this relati immve unogenici asstyay under the conditions tested (see Figures 15-16; Tables 14-16). This Shiga toxin effector scaffold compris ed(1) five, disrupted epitope regions, two of whic hinvolv multied ple amino acid residue substitutions, and (2) an endogenous epitope region disrupt edby an embedded, heterologous T- , cell epitope In. the second study, the combination, de-immunize protd, eas e- cleava resge ista Shigant, toxin effecto scar ffol SLT-d 1 A-combo 16 appeared to be more de-immuniz edthan the combination, de-immunize protead, se-clea vage resistant, Shiga toxin effector scaffold SLT-lA-combol9 esp, eciall at earliery time- point (sees Figur e15-pane B;l Table 14). In the fourth study, the combination, de- immunized, protease-cleava resistage Shigant, toxin effector scaffold SLT-1 A- combo 1 appeared to be the leas de-imt munized of the tested, Shiga toxin effector polypepti desSLT-1 A-combos (1, 10, 12 and 15) (see Figur e16-paenl A; Table 16).
In the four thstudy, the combination, de-immunize proteased, -clea resistvageant, Shiga toxin effector scaffold SLT-1 A-combo 15 appeared to be more de-immunize d than SLT-lA-combol2, especiall at latey time-pointsr (,see Figure 16-pane X;l Table 16). 838. 838. 838. id="p-838" id="p-838" id="p-838" id="p-838" id="p-838" id="p-838" id="p-838" id="p-838" id="p-838" id="p-838"
[838] The results of thes imme unogenicit studiesy indica thatte two types of epitope region disruptio aminons, acid residue substitutions and embedded, heterologous CD8+, T-cell epitope cans, contribute to the de-immunization of a Shiga, toxin effector polypeptide. Further the, combinati ofon both types of disruptions in the same Shiga toxin effector polypeptide can result in a more de- immunized, Shiga toxin effector polypeptide, lire overall magnitude of antibody induction in the mice in the groups administered exemplar cytotoxic,y', cell-targeting molecules comprising thes combie natio de-imn, munize CD8+d, T-cell hyper- immunized, protease-cleava resistage Shigant, toxin effector polypeptides was reduced as compared to magnitude of antibody induction in the mice in the group administer theed cell-target moleculeing comprisin theg protease-cle avageresist ant but otherwise wild-type Shiga toxin effector polypeptide region. The decreas ines the ELISA signal value ofs the cell-target moleculeing comprs isin certaig n,WO 2016/196344 PCT/US2016/034778 328 combination de-i, mmunize proteasd, e-clea resvageista Shigant, toxin effector scaffolds demonstr thatates these particula scaffoldsr were successfully de- immunized (z.e. had reduc edimmunogeni potentiac in maml mal s).Thus, the Shiga toxin effector polypepti desSLT-lA-combol, SLT-lA-combo7, SLT-lA-combol 0, SLT-lA-combol2, SLT-lA-combol5, SLT-lA-combol 6, and SLT-lA-combol9 exhibite reducedd immunogeni potentiac in mammall ands, any, exemplar cell-y, targeting molecule comprisi thesng polypeptie desshould be de-immunized as compared to an analogou moles cule comprisin onlyg wild-type or merely proteas e- cleava resge ista Shigant, toxin effector polypeptides due to furin-cleavage motif mutations in the native positily oned regio fromn amino acid residues 238 to 257 in Six A and SLT-1 A. 839. 839. 839. id="p-839" id="p-839" id="p-839" id="p-839" id="p-839" id="p-839" id="p-839" id="p-839" id="p-839" id="p-839"
[839] Differences observe ind the immunogenic potentials of the different combination de-immunized,, protease-cleava resistage Shigant, toxin effector polypepti destested shows that certa combinatin ions of de-immuniz edsub-regions and/o embedder T-celd epitopel sub-regions reduce immunogenicit by ydifferent magnitudes. For example in study 2, SLT-1 A-combo 10 was more de-immunize d than SLT-lA-combol 6 and SLT-lA-combo 19, especia llyat later time-points (Tabl e14; Figur e15-panel B), despite combol andb combo! comprising9 more disrupt edepitope regions and total amino acid residue substitutions compar toed a wild-type Shiga toxin A Subunit than SLT-1 A-combo 10 compris (Tablees 8). In study 4, SLT-1 A-combo 10 was more de-immunized than SLT-1 A-combo 12, especially at later time-points (Tabl e16; Figure 16-pane A)l despite that both SLT- lA-combol2 and SLT-1 A-combo 15 comprise more disrupt edepitope regions and total ammo acid residue substitutions compared to a wild-type Shiga toxin A Subunit than SLT-lA-combolO compris (Tables e8). Thus, the cumulati ve combinati ofon additional B-cell epitope region disruptions did not necessa rilyresult m additiona decrl eas ines immunogenicit buty rather may resul int increa sein s unwante immunogenicd itie suchs, as, e.g., at later time-points (see Figure 15-panel B and compare the results for SLT-1 A-combo 10 with the results for SLT-1A- combolb and SLT-lA-combol9).WO 2016/196344 PCT/US2016/034778 329 I. Testing the Ability of Exempla ry,Cell-Target Moleculing toes Deliver a T-Cell Epitope-Peptide to the MHC Clas 1s Pathw ayof a Cell for Presentation 840. 840. 840. id="p-840" id="p-840" id="p-840" id="p-840" id="p-840" id="p-840" id="p-840" id="p-840" id="p-840" id="p-840"
[840] The presentat ofion a T-cell epitope by the MHC clas I ssystem targets the presenting cell for killing by CTL-mediated lysi ands als triggero immunes stimulat inion the loca arel a.By engineering cell-target molecing ules comprisi ng Shiga toxin effector polypepti descomprisin heteg rologous immuno, geni epitopesc , the targeted delive andry presentat ofion immuno-stimulator antigensy may be accomplishe Hied. presenta tionof immuno-stimulatory non-self antige ns,such as e.g. known viral antigens with high immunogenic byity, targe cellst signal tos other immune cells to destroy the targe cellst as wel asl to recruit more immune cells to that area within an organism. 841. 841. 841. id="p-841" id="p-841" id="p-841" id="p-841" id="p-841" id="p-841" id="p-841" id="p-841" id="p-841" id="p-841"
[841] In order to simultaneously de-immunize and provide for T-cell epitop e presentation on the targe cetll surfac wite hin the same Shiga toxin effector polypeptide region, a predicted B-cell epitope regio wasn disrupte byd replacing amino acid residues within it with an immunogenic T-cell epitope regio predicn ted to bind to huma nMHC class I molecules. 842. 842. 842. id="p-842" id="p-842" id="p-842" id="p-842" id="p-842" id="p-842" id="p-842" id="p-842" id="p-842" id="p-842"
[842] In this Example the, abilities of exempla ceryll-targeti moleculeng ofs the present invention to deliv erT-cell epitopes to the MHC class I pathway of targ et cells for presentation to the targe ceilt surfa ceare investigate In d.addition, the functiona consequencesl of targe celt ’ls MHC clas I spresenta tionof T-cell epitopes delivered by exemplar cell-y target molecing ules of the prese ntinvention are investigate by obsed rving various immune responses induced by the presentation of tiie delivered epitope-pept byide an MHC I molecule. 1. Testing the Ability of a Molecul toe Deliver a T-Cel lEpitope-Pepti tode the MHC Clas Is Pathw ayfor Presentati onon the Cell Surface 843. 843. 843. id="p-843" id="p-843" id="p-843" id="p-843" id="p-843" id="p-843" id="p-843" id="p-843" id="p-843" id="p-843"
[843] Routine assa ysknown in the art are used to investiga thete ability of exemplar moleculey ofs the prese ntinvention to deliv era T-cell epitope to a MHC class I molecule (see e.g. WO 2015/113007). In particula a flowr, 7 cytome־ trymethod is used to demonstrat delivere andy extracellular display of a T-cell epitope-peptide (inserted or embedde ind a Shiga toxin effecto polypeptider in comp) lex ׳with MHC class I molecules on the surfaces of targe celt ls. This flow cytome7 trymethod utilizes soluble huma nT-cell receptor (TCR) multimer reagents (Soluble T-CellWO 2016/196344 PCT/US2016/034778 330 Antigen Receptor STAR™ Multimer Aitor, Bioscience Corp., Mirama FL,r, U.S.), eac hwit hhigh-affin bindinity tog a different epitope-huma HLAn complex. 844. 844. 844. id="p-844" id="p-844" id="p-844" id="p-844" id="p-844" id="p-844" id="p-844" id="p-844" id="p-844" id="p-844"
[844] Each STAR™ ICR multimer reagent is derived from a specif icT-cell receptor and allows detection of a specifi peptide-c MHC comple basex don the abilit ofy the chosen TCR to recogniz a specife icpeptide presented in the context of a particular MHC class I molecule These. TCR multimers are compos edof recombina humannt TCRs which have been biotinylated and multimeriz withed streptavidin. The TCR multimers are labeled with phycoerythr (PE).in Thes eTCR multimer reagents allow the detection of specif icpeptide-MHC class 1 complexes presente ond the surface ofs human cel lsbecause each soluble TCR multimer type recogniz andes stab lybinds to a specif icpeptide-MHC comple underx varied conditions (Zhu X et al., J Immunol 176: 3223-32 (2006)). Thes eTCR multime r reagents allow the identification and quantita tionby flow' cytometr of peptide-y MHC cla ssI complexe press ent on the surfaces of cells. 845. 845. 845. id="p-845" id="p-845" id="p-845" id="p-845" id="p-845" id="p-845" id="p-845" id="p-845" id="p-845" id="p-845"
[845] The targ cellset used in this Example are availa blefrom the ATCC (Manassas VA, U.S.), National Cance Instir tute of the U.S. (Frederic MD,k, U.S.), and/o DSZMr (Braunschweig, DE). Using standar flowd cytometr methy ods known in the art, the target cells are confirm toed express on thei celr surfal ces both the appropriate MHC-cla ssI molecule and the extracellula targe rbiomolecult ofe the cell-targetm moietyg of the cell-targetin moleculeg useds m this Example. 846. 846. 846. id="p-846" id="p-846" id="p-846" id="p-846" id="p-846" id="p-846" id="p-846" id="p-846" id="p-846" id="p-846"
[846] The targe cellst are treated with exempla ceryll-targeti molecng ules of the present invention that each comprise a Shiga toxin effector polypept idecomprising an embedded or inserted T-cell epitope. Certa exemin plary molecule ofs the prese nt invention teste ind this Example are catalytica impallyire ord inactivate by thed addition of one or both of the following mutations Y77S: and El 67D. Sets of target cel lsare treated by exogenou adminis stration of the differ entexemplary cell - targeting molecule ofs the invention at concentra tionssimilar to those used by others taking into accou ntcell-type specif icsensitivit toies Shiga toxins (see e.g. Noakes K et al., FEES Lett 453: 95-9 (1999)). The treated cells are then incubated for six hour ins standar conditid ons, including at 37°C and an atmosphere wit h5% carbon dioxide, to allow' for intoxica tionmediated by a Shiga toxin effector polypept ide region Then. the cells are washed with cell culture medium, re-suspende in dfresh cell culture medium, and incubated for 20 hour priors to staining with the WO 2016/196344 PCT/US2016/034778 331 appropriate STAR™ multimer reagent. Additional time-points and setup conditions are also tested. 847. 847. 847. id="p-847" id="p-847" id="p-847" id="p-847" id="p-847" id="p-847" id="p-847" id="p-847" id="p-847" id="p-847"
[847] As contro setsls, of targ celet lsare treated in three conditions: 1) without any treatment ("untreated'’) meaning that no exogenous molecules are added, 2) with exogenously administer controled antigen-pepti andde, 3) with exogenously administere controld antigen-pept combineide withd a Peptide Loading Enhancer (‘TLE," Aitor Bioscienc Corp.,e Mirama FL,r, U.S.). The contr antigen-ol peptide peptide combine withd PLE treatmen allowedt for exogenous peptide loading and served as a positive contro Celll. displs aying the appropriate MHC clas I shaplotype can be forc edto load the appropri ateexogenously applied peptide from an extracellular space (i.e. in the absenc ofe cellular internalization of the applied peptide or) in the presence of PLE, which is a mixture of B2-microglobuli andn other components. 848. 848. 848. id="p-848" id="p-848" id="p-848" id="p-848" id="p-848" id="p-848" id="p-848" id="p-848" id="p-848" id="p-848"
[848] After the treatments all the, sets of cells are washed and incubated wild the appropriate STAR multimer reage fornt one hour on ice . Ure cells are washed and tire fluorescen ofce the samples are measure byd flow' cytometry using an Accuri™ C6 flow7 cytomet (BDer Bioscienc esSan, Jose ,CA, U.S.) to detec thet presence of and quantif anyy STAR™ multimer bound to cel lsin the population (sometime s referred to herein as "staining"). 849. 849. 849. id="p-849" id="p-849" id="p-849" id="p-849" id="p-849" id="p-849" id="p-849" id="p-849" id="p-849" id="p-849"
[849] The untreat contred isol used to identify the positive and negat ivecel l populations by employing a gate w hich results in les thans 1% of cells from the untrea tedcontro in lthe "positive" gate (representing background signa l).Ure same gate is then applied to the oilier samples to characteriz tire posite ive population for eac hsample. 850. 850. 850. id="p-850" id="p-850" id="p-850" id="p-850" id="p-850" id="p-850" id="p-850" id="p-850" id="p-850" id="p-850"
[850] The detection of the exogenously administe red,embedded or insert T-ceed ll epitope complexed with human MHC clas Is molecules on the cell surface of intoxicat targed cellset demonstr thatates cell-targetin moleculeg comprs isin theg embedde ord inserted T-cell epitope-pept areide capable of entering targe cellst , performi sufngficient sub-cellula routr ing, and delivering enough T-cell epitope to tire MHC class I pathway for surfa cepresentation on the targe cellt surface.WO 2016/196344 PCT/US2016/034778 332 2. Testing the Ability of a Molecule to Induce Cytoto xicT-Cell Mediated Cytolys is of Target Cell ands Other Immune Responses 851. 851. 851. id="p-851" id="p-851" id="p-851" id="p-851" id="p-851" id="p-851" id="p-851" id="p-851" id="p-851" id="p-851"
[851] Routine assa ysknown in the art are used to investiga thete functional consequences of target cel’ls MHC class I presenta tionof T-cell epitopes delivered by exempla7 cellry -targeting molecules of the invention (see e.g. WO 2015/113007).
The functional consequen toces investiga includete CTL activat ion,CTL-mediated target-cell-killing, and cytokine release by CTLs. 852. 852. 852. id="p-852" id="p-852" id="p-852" id="p-852" id="p-852" id="p-852" id="p-852" id="p-852" id="p-852" id="p-852"
[852] A CTL-base dcytotoxic assaity isy used to assess the consequences of epitope presentation. The assa involvesy tissue-cultured targe cellst and T-cells .
Target cel lsare intoxicated as described in WO 2015/113007. Briefly, targe cellst are incubated for six hour ins standar conditionsd wit hdiffer entexogenous ly administe red,cell-target moleingcule wheres, certain cell-targeting molecules comprise a Shiga toxin effector polypept ideof tire invention. Next, CTLs are added to the intoxicated targ cellset and incubated to allow for the T-cel lsto recognize and bind any target-ce displayinglls epitope-peptide/MHC class I complexes. Then certain functiona consequencesl are investigate usingd standar methd ods known to the skilled worker, including CTL bindin tog target cells target, -cell-killing by CTL- mediated cytolysi ands, the release of cytokines, such as interfer gammaon or interleukins by ELISA or ELIspot. 853. 853. 853. id="p-853" id="p-853" id="p-853" id="p-853" id="p-853" id="p-853" id="p-853" id="p-853" id="p-853" id="p-853"
[853] The activati ofon CTLs by targe cellst displaying epitope-peptide/MHC cla ss I complexe is squantified using commerciall availay bleCTL response assays e.g., CytoTox96® non-radioa ctiassave ys(Promega Corp., Madison, WI, U.S.), Granzyme B ELISpot assa ys(Mabtech, Inc., Cincinnati, OH, U.S.), caspase activity assays, and LAMP-1 transloca flowtion cytomet assays.ric To specifically monitor CTL-mediated killing of targe celt ls, carboxyfluores succinimidylcein ester (CFSE) is used to target-ce forlls hi vitro and in vivo investigat asion described in the art (see e.g. Durwar Md et al., J Vis Exp 45 pii 2250 (2010)).
Summan7 of Exampl 2e 854. 854. 854. id="p-854" id="p-854" id="p-854" id="p-854" id="p-854" id="p-854" id="p-854" id="p-854" id="p-854" id="p-854"
[854] Exemplary, cell-target molecing ules comprisin combig natio Shigan, toxin effector polypeptide with multiple B-cell epitope regio disruptn ions wer ede- immunized as shown by reductions in both antigenicity7 and immunogenicit as y compared to reference molecules. In addition, this Example shows that certa de-in, immunized cell-targeti molecng ules comprisin combination,g Shiga toxin effector WO 2016/196344 PCT/US2016/034778 333 polypepti descomprisin multg iple B-cell epitope regio disruptionsn furi, ncleava ge motif disruptions and/or, embedded T-cell epitopes retain at a significant leve ofl one or more Shiga toxin effecto functions,r such as, e.g., catalytic ribosome inhibition, intracell routularing, and cytotoxicity. 855. 855. 855. id="p-855" id="p-855" id="p-855" id="p-855" id="p-855" id="p-855" id="p-855" id="p-855" id="p-855" id="p-855"
[855] Table 17 summariz esresults from Exampl 2e for the exemplary, protease- cleava resistge ant, de-immunize cell-d, target molecing ules of the prese ntinvention which, comprise the Shiga toxin effector polypeptide SLT-lA-combol, SLT-1A- combo?, SLT-lA-combolO, SLT-lA-combol2, SLT-lA-combol5, SLT-1A- combol6, or SLT-lA-combol9.
Table 17. Summary of Exempla Celry l-Target Molecuing les Empiricall Testy ed for Cytotoxici andty Reduced Immunogen Poteic ntia in Maml mals Cytotoxicity Immunogenic ity cell-target ing (CD50 in nM) compared to SET- 1A-FR: scFv-1 molecul scaffole d Varies with the cell-line SLT-1A- 0.02 41 - 99 % combol ::scFv-1 SLT-1A- 0.01, 0.02, 0.03, 0.12, 0.13, 0.39 4 - 36 % combo? ::scFv-1 SLT-1A- 0.03, 0.07, 0.08, 0.25, 0.84 1 - 43 % combo 10: :scFv-1 SLT-1A- 0.07, 0.18 6 - 70 % combo 2:l :scFv-1 SLT-1A- 0.05, 0.09 3 - 37 % combol5::scFv-l SLT-1A- 0.03, 0.12 14 - 72 % combol :scFb: v-1 SLT-1A- 0.03, 0.16 64 - 107 % combo 19: :scFv-1 SLT-1A-FR 0.01, 0.02, 0.05, 0.10, 0.28 100 % ::scFv-1 856. 856. 856. id="p-856" id="p-856" id="p-856" id="p-856" id="p-856" id="p-856" id="p-856" id="p-856" id="p-856" id="p-856"
[856] The exempla ry,protease-cle avageresistant, de-immunize celld, -targeting molecules of the invention shown in Table 17 all exhibited significa levelnt ofs cytotoxici whichty were comparable to cell-target molecing ules comprisin wild-g type Shiga toxin Al fragments. Thes eexemplary, protease-cle avageresistant, de- immunized, cell-targetin moleculesg of the invention exhibit catalyti inhibitionc of transla bytion ribosomes intrac, ellular routing comparable to wild-type a Shiga toxin A Subunit and/o Alr fragment, and cytotoxicity comparable to a cell-targeting molecules comprising a wild-type Shiga toxin Al fragment and/o Ar Subunit.
Thes eexemplar proteasy', e-cle avageresistant, de-immunize celld, -targetin gWO 2016/196344 PCT/US2016/034778 334 molecules of die invention exhibit levels of catalyti activc itycomparable to a wild- type, Shiga toxin A Subunit and/or Al fragment. These exemplary, proteas e- cleava resge ista de-immunized,nt, cell-target moleculeing ofs the invention exhibi t levels of cytotoxici compaty rable to a. wild-type Shiga, toxin A Subunit and/or Al fragment. Thes eceil-target molecing ules of the invention all exhibite reducedd immunogenicit in mamy mal ass compared to cell-targetin molecg ules comprisin a g Shiga toxin effector polypeptide region consist ingof (1) a wild-type Shiga, toxin A1 fragment and/or (2) the furin-cleavage resistant, Shiga toxin effector polypept ide SLT-1A-FR. Furthermor certae, cell-in target molecing ules of the present invention exhibit increas staed bilit improvedy, in vivo tolerability, and/o ther ability to deliver heterologous T-ce,ll epitopes for MHC cla ssI presentat byion targe celt ls. 857. 857. 857. id="p-857" id="p-857" id="p-857" id="p-857" id="p-857" id="p-857" id="p-857" id="p-857" id="p-857" id="p-857"
[857] The results shown in Exampl 2e reinfor thece idea that various exe, mplar y, epitope region disruptions may be combine togetherd in a single molecule to crea te great reducter ions in antigenicit and/oy immunogenicir whilety still retaining significant levels of one or more Shiga, toxin effector function (see e.g. WO 2015/113005) and sometimes providing another functiona fealture not(s) present in wild-type Shiga toxin A Subunits such, as, e.g., furin-cleavage resista nceand/or the ability to deliv era heterologous T-cell, epitope to a targe cellt Certain,. combination de-im, munize d,Shiga toxin A Subunit effecto polypeptidesr of the present invention exhibi synergistict reductions in immunogenicit as comparedy to the sum. of thei partiar de-imlly munized sub-region suchs, as, e.g., SLT-lA-combo7 SLT-lA-combolO, and SLT-lA-combol5. 858. 858. 858. id="p-858" id="p-858" id="p-858" id="p-858" id="p-858" id="p-858" id="p-858" id="p-858" id="p-858" id="p-858"
[858] The followi substitutionsng have been made and tested in at leas onet Shiga toxin effector polypeptide which retaine a signifd icant leve ofl in vitro, ribosome inhibiti onand/o cytotoxicr KIA,ity: KIM, T4I, S8I, T9I, KILA, S33I, S33C, S43N, G44L, S45V, T45V, S45I, T45I, G46P, D47M, N48V, L49A, F50T, A51V, D53A, D53N, V54L, V54I, R55A, R55V, R55L, G56P, 157F, I57M, D58A, D58V, D58F, P59A, P59F, E60I, E60T, E60R, E61A, EM Y. E61L, G62A, D94A, S96I, T104N, A105L, T107P, L108M, S109V, G110A, D111T, D141A, V154A, G147A, T180G, T181I, D183A, D183G, D184A, D184A, D184F, L185D, S186A, S186F, G187A, G187T, R188A, S189A, D198A, R205A, C242S, R248A, and R251A. 859. 859. 859. id="p-859" id="p-859" id="p-859" id="p-859" id="p-859" id="p-859" id="p-859" id="p-859" id="p-859" id="p-859"
[859] Despite the challenges predicting successfill substitutions a priori, the data , provided in the Examples herein give reasons to believe that certa aminoin aci d substitutions are likely to successfully reduce antigenicit and/oy immunoger nicity WO 2016/196344 PCT/US2016/034778 335 while maintaining significant Shiga toxin effector fimction(s). The term "successful" is used here to mea none or more amino acid residue substitutions in a predicted epitope region resulted in a Shiga toxin effector polypeptide whic h retaine oned or more Shiga toxin effector functions. For example, substituti onsat specif icamino acid position showns herein as successfully tolerating substitutions are likely to be successful for retaini atng lea stone Shiga toxin effector function when substituted wit hcertai othen amir no acids. Successful single amino aci d substitut ionmay generally be combine withd othe sucr cessful amino acid substitutions in a different epitope region to generat de-ime mtmized, Shiga toxin effector polypeptides which reta signifin icant Shiga toxin effecto functr ion( s).
Similarly, the demonstrati thaton cell-target moleculesing comprising Shiga toxin effector polypeptide regions with multiple single amino acid substitutions within the same epitope region retaine enzymatd actic ivity suggests that successful single amino acid substituti inon the same epitope region may generally be combine withd othe singler amino aci dsubstitutions in the same epitope regio ton genera de-te immunized, Shiga toxin effector polypeptides which reta signifin icant Shiga toxin effector function(s). 860. 860. 860. id="p-860" id="p-860" id="p-860" id="p-860" id="p-860" id="p-860" id="p-860" id="p-860" id="p-860" id="p-860"
[860] It has been empiricall demonstry atedthat certai substitutionsn (KIA, KIM, T4I, SSI, T9I, K11A, S33I, S33C, S43N, G44L, S45V, T45V, S45I, T45I, G46P, D47M, N48V, L49A, F50T, A51V, D53A, D53N, V54L, V54I, R55A, R55V, R55L, G56P, I57F, 157M, D58A, D58V, D58F, P59A, P59F, E60I, E60T, E60R, E61 A, E61V, E61L, G62A, D94A, S96I, T104N, A105L, T107P, L108M, S109V, G110A, Dll IT, D141A, G147A, V154A, G147A, T180G, T181I, D183A, D183G, D184A, D184A, D184F, L185D, S186A, S186F, G187A, G187T, R188A, S189A, D198A, R205A, C242S, R248A, R251A, and/o combir natio thereof)ns and certain position tolers ated substitutions (1, 4, 8, 9, 11, 33, 43, 44, 45, 46, 47, 48, 49, 50, 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 94, 96, 104, 105, 107, 108, 109, 110, 111, 141, 147, 154, 180, 181, 183, 184, 185, 186, 187, 188, 189, 198, 205, 242, 248, and 251) while retaini ang significant level of activity for at lea stone Shiga toxin effector function. This empiric dataal suggest certa othein epitr ope disrupting substitutions and combinations of epitope disrupting substituti onswhich may be used to generate de-immunize Shigad, toxin effecto polypeptidesr whic hreta signiin fica Shigant toxin effector function(s). It is predictable that othe aminor acid substitutions to amino acid residues of a conserva functionaltive group will also be tolerate Ford. WO 2016/196344 PCT/US2016/034778 336 example, othe substitutir onsknown to the skilled worker to be simila tor any of KIA, KIM, T4I, SSI, T9I, K11A, S33I, S33C, S43N, G44L, S45V, T45V, S45I, T45I, G46P, D47M, N48V, L49A, F50T, A51V, D53A, D53N, V54L, V54I, R55A, R55V, R55L, G56P, I57F, I57M, D58A, D58V, D58F, P59A, P59F, E60I, E60T, E60R, E61A, E61V, E61L, G62A, D94A, S96I, T104N, A105L, T107P, L108M, S109V, G110A, Dll FT, D14LA, G147A, V154A, G147A, T180G, T1811, D183A, D183G, D184A, D184A, D184F, L185D, S186A, S186F, G187A, G187T, R188A, S189A, D198A, R205A, C242S, R248A, or R251 A will als beo able to disrupt an epitope while maintaining at least one Shiga toxin effector function.
Exampl 3.e Furin-Cleava Resigesta nt,Shiga Toxin A Subunit Effecto r Polypeptid andes Cell-Targeti Moleculng Comprises ing the Same 861. 861. 861. id="p-861" id="p-861" id="p-861" id="p-861" id="p-861" id="p-861" id="p-861" id="p-861" id="p-861" id="p-861"
[861] Furin-cleavage resistant, Shiga toxin A Subunit effector polypeptides were creat anded tested as compone ntsof cell-targetin molecg ules wherei eachn cell- targeting molecule comprised a cell-targe immuting,noglobulin-ty bindingpe, region.
To enginee protear resse ista nceinto a Shiga toxin effector polypepti twode, amino acid residue substitutions, R248A and R251 A, wer introducede into Shiga toxin effector polypeptides as described in Example 2. The Shiga toxin effector polypept ideSLT-1A-FR (SEQ ID NO:5) was used to create the ceil-targetin g molecule SLT-lA-FR::scFv- (SEQ9 ID NO:41).
A. Quantifying Furin Cleavage of a. Molecule of the Present Inventi onRelative to a Reference Molecule 862. 862. 862. id="p-862" id="p-862" id="p-862" id="p-862" id="p-862" id="p-862" id="p-862" id="p-862" id="p-862" id="p-862"
[862] lire cell-targetin molegcule SLT-lA-FR::scFv-9 (SEQ ID NO:41) was test ed using an in vitro, furin-cleavage assay to quanti fyfuri ncleava asge compared to a wild-type contr usingol methods known to the skilled worker (see e.g. WO 2015/191764). To assess the ability of furin to clea veSLT-lA-FR::scFv-9, purified protei samn ples in phosphate buffer edsaline (PBS) wer eincubat edwith furin (New England Biolabs, Ipswich, MA, U.S.) at 0.5 furin activit unitsy (U) per microgra m (pg) of sample protein in furi ncleavage buffer (100 millimola (mMr) HEPES (4-(2- hydroxyethyl)-!-piperazineethan acid),esulf pH 7,onic 1 mM CaCh) for 30 hours at °C. Control samples wer eincubated without furin at 4°C or 30°C in the same buffe r.The various cell, -targetin molegcule samples wer eelectrophorese on SDS,d WO 2016/196344 PCT/US2016/034778 337 polyacrylamide gels under denaturing conditions and stained with Coomass ie (Figur e17). 863. 863. 863. id="p-863" id="p-863" id="p-863" id="p-863" id="p-863" id="p-863" id="p-863" id="p-863" id="p-863" id="p-863"
[863] Figure 17 shows a pictur ofe a gel with the lane numbes red and a figure legend indicating which lane was load edwit hwhich sampl e:either a cell-targeting molecule comprisin a gwild-type Shiga toxin effector polypeptide region (SLT-1A- WT) or a furin-cleava sitege disrupted, Shiga toxin effecto polypeptr ide(SLT-1A- FR). 'The lane markeds "MW Mark"er show the migration patte ofrn a protein molecular weight ladder along with the approxim atesize of individual ladder protein band ins kiloDaltons (kDa) for use as an internal molecular weight refere ncethat allows for the estimatio of nthe sizes of proteins in the numbered lanes The. figur e legend indicat thees pre-treatme condint tions of the cell-targeti molengcule sampl es with the temperature in degree Celss ius (°C), duration, and whether any furin was added by denoting the amount of furin activity units per microgram (labele "U/pgd furin ")or "no furin" for zero units. 864. 864. 864. id="p-864" id="p-864" id="p-864" id="p-864" id="p-864" id="p-864" id="p-864" id="p-864" id="p-864" id="p-864"
[864] Figure 17 shows that SLT-1 A-FR::scFv- 9(SEQ ID NO:41) was resistant to proteolytic cleavage by human furin. The cell-targetin molecg ules test edin this assay wer eboth about 56 kDa in size and compris eda Shiga toxin effector polypept ideof about 28 kDa (identic inal size for both SLT-1 A-WT or SLT-1A-FR) linked to a carboxy-termina linker land binding region which togeth werer eabou t28 kDa in size. If furin cleavage had occurred in the surfa ceexposed, extende loopd 242-251 of SLT-1 A, then the expecte resuld wouldt be two protein band withs near equal molecul weightsar of around 28 kDa each. If furin cleavage occurs precisely at the carboxy peptide bond of the arginine at position 251 of the WT scaffold in SLT-1 A-WT: :scFv-9, then the two resulting protein bands should have the molecul weightsar 0f27.5 kDa for SLT-1A (either WT or FR) and 0f28.3 kDa for scFv-9. 865. 865. 865. id="p-865" id="p-865" id="p-865" id="p-865" id="p-865" id="p-865" id="p-865" id="p-865" id="p-865" id="p-865"
[865] The gel was analyzed using GelAnalyzer 2010 softwa re.This software detecte thed lane ofs the gel pictur edin Figur e17 and the band withins each lane .
Using background subtracti mode,on the background was automatica definedlly and subtrac tedfrom the volume ofs all band anals yzed using a rolling bail background subtracti witon hthe ball radius set at 25. The results of this quantitative band analysis of the gel pictur edin Figure 17 is summariz edin Table 18. Table 18 shows the relat ivemobility and raw volume values for certain band migras ting around 56 kDa in lane 1-6s in the gel pictured in Figur e17. The "no furin4° ’־C treatment of WO 2016/196344 PCT/US2016/034778 338 tiie cell-targete molecd ule (SLT-lA-WT::scFv- 9)(SEQ ID NO:42), which compris aes wild-type Shiga toxin effector polypepti resude, lts in lane #2 was used as a control to determi thene percentage of uncleav mateed rial at 30°C in the absenc ore presence of furin (lane #2s and #3, respectively). For the SLT-1A- FR::scFv-9 samples, the "no furin" 4°C lane (lane #4) was used to determine the percenta ofge uncleaved material at 30°C in the absence or presence of furi n(lanes #5 and #6, respectively) For .each molecule, the "percenta unclege aved" of the ~56 kDa band for eac hfurin treated sample was calculated by the followi formulang ; (Raw Volum eof-56 kDa sample band) / (Raw Volum eof -56 kDa band from the "no lurm " 4°C treatment) x 100 (see Table 18).
Table 18. Quantificati ofon Furin-Cleava Relage tive to a Reference Molecule Lane Relative mobility (Rf) Percenta ge # Treatment of ~ 56 kDa Band Raw7 Volume uncleaved SLT-lA-WT::scFv-9 no furin, 4°C 1 0.39 753 100.0 % no furin, 30°C 2 0.36 766 101.7 % 0.5 U/ug furin, 3 0.34 246 32.7 % °C ' SLT-lA-FR::scFv-9 no furin, 4°C 4 0.37 995 100.0 % no furin, 30°C 5 0.39 985 99.0 % 0.5 U/ug furin, 6 0.37 1000 100.5 °C ' 866. 866. 866. id="p-866" id="p-866" id="p-866" id="p-866" id="p-866" id="p-866" id="p-866" id="p-866" id="p-866" id="p-866"
[866] For the wild-type furin site samples, the amount of protei inn the - 56 kDa band for the sample incubated wit hfurin was reduced compared to the amount of protei inn the - 56 kDa band for "no furin" samples (Tabl e18; Figur e17, lane #3 compared to lane #1s and #2). The furin treatment of SLT-lA-WT::scFv-9 resulted in the producti ofon two new band ofs about 28 kDa (Figur e17, lane # 3), which match the expected size sof furin-cleava productsge resulting from cleavage at the carboxy-termi ofnus the Shia toxin Al fragment region of SLT-lA-WT;:scFv-9.
For the SLT-lA-FR::scFv- sampl9 es, the amount of protein in the ~ 55 kDa band in the sample incubated with furin lane appeared unchange fromd the amount of protei inn the - 55 kDa band for "no furin" samples (Tabl e18; Figur e17, lane #6 compared to lane #4s and #5). 867. 867. 867. id="p-867" id="p-867" id="p-867" id="p-867" id="p-867" id="p-867" id="p-867" id="p-867" id="p-867" id="p-867"
[867] This quantitat analive ysis showed that a cell-targetin moleculeg designed with the wild-type Shiga toxin effector polypept ideSLT-1A-WT and a carboxy- termina bindingl regio exhibiten aboutd 67.3% cleavage with about 32.7% of SLT- WO 2016/196344 PCT/US2016/034778 339 1A-WT::scFv-9 remaining uncleav ed.Tire percenta ofge furin-cleavage as compared to a refere ncemolecule can be express ased a ratio of [(available material - uncleave / d)availa blematerial] of the molecule of interest to [(available materia - l uncleaved)/avail materiaable ofl] the refere ncemolecule In. this assay, SLT-1A- FR::scFv- 9exhibite [(985-1000)71000]d / [(766-246)7766] = -1.5 % cleavage of the refere nceor approximat zeroely cleavage. 868. 868. 868. id="p-868" id="p-868" id="p-868" id="p-868" id="p-868" id="p-868" id="p-868" id="p-868" id="p-868" id="p-868"
[868] 'This assay showed that a cell-target moleingcule designed with the Shiga, toxin effector polypeptide SLT-1A-FR exhibite 0%d cleavage wit h100% of the ceil- targeting molecule remaining uncleaved. Thus, the SLT-1A-FR scaffold appear tos be resistant to furin cleavage in this assay under the conditions tested.
B, Testing the In Vivo Tolerabi litof Cell-y Target Molecing ules of the Present Invention Using Laboratory Animals 869. 869. 869. id="p-869" id="p-869" id="p-869" id="p-869" id="p-869" id="p-869" id="p-869" id="p-869" id="p-869" id="p-869"
[869] The in vivo tolerabili of tyexemplar cey,ll-targeti moleculeng ofs the present invention is test edusing mice in order to determine the degree to which overt adverse effects wer detece ted at various dosages of cell-target moleingcule samples .
The tolerabili studiety ares performed, using method knowns to the skill edworker and/o descrr ibed herein (see e.g. WO 2015/191764). For example, mice are inject ed with cell-targeti molecng ule samples or vehicl controlse at doses ranging from 0.25 to 5.00 milligra perms kilogram of body weight per injection (mg/kg/inj) for three times a wee kover severa weeksl In. order to assess in vivo tolerability, the injecte d mice are monitored for changes in health and clinic signs,al such as, e.g., aspect ofs morbidity, morbundity, body weight, physical appearance, measureable clinic al signs, unprovoked behavior, and response to sexternal stimuli, such as, (see e.g.
Morton D, Griffit P,hs Vet Rec 116: 431-43 (1985); Montgomery' C, Cancer Bull 42: 230-7 (1990); Ullman-Culle M,re Foltz C, Lab Anim Sc 49; 319-23 (1999); Clingerman K, Summer L,s J Am Assoc Lab Aram Sci 51: 31-6 (2012)). Euthanas ia may be used in respons toe signs of m orbidity and/or morbundit and,y thus, creat a e mortality time-point For. example, a. decreas in ebody weight of 15-20% in 2-3 days can be used as a sign of morbidity in rodents and as a justification for euthanization (see e.g. Institute of Laboratory Animal Research 2011. Guide for the care and use of laboratory animals, 8 th ed., Washingt on,DC, U.S.: National Academi esPress).WO 2016/196344 PCT/US2016/034778 340 870. 870. 870. id="p-870" id="p-870" id="p-870" id="p-870" id="p-870" id="p-870" id="p-870" id="p-870" id="p-870" id="p-870"
[870] The cell-targetin molecg ules of the prese ntinvention that compris fure in- cleava resge ista Shigant, toxin effector polypeptides exhibit improved tolerability (e.g. improved non-specifi toxicc ityprofil es)as compar toed relate refd, ere nce molecules comprising wild-type Shiga toxin Al fragmen (seets e.g. WO 2015/191764). Such improved in vivo tolerability may be due to the increas ed stability of a linkage between a Shiga toxin effector polypept ideand a binding region and/or toxic componen oft the cell-targeti molecng ule.
C. Testing the Targete Cytotoxicityd 7 arid Efficacy of Exempla ry,Cell-Targeting Molecul ofes the Present Invention 2/1 Vivo Using Anima Modelsl 871. 871. 871. id="p-871" id="p-871" id="p-871" id="p-871" id="p-871" id="p-871" id="p-871" id="p-871" id="p-871" id="p-871"
[871] Animal models are used to determine the in vivo effect ofs exempla7, ryShiga, toxin effector polypepti descombo(n) and cell-targeti molecng ules comprisi theng aforementioned to targe positive,t neoplas cellstic Variou. mices strains are used to tes thet effects of the cell-targeting molecule ons xenograf tumorst in mice after intravenous administration of eac hmolecule to those mice. In certain experiments, a disseminate xenografd modelt for huma ntumors is used to determi thene in vi vo efficacy of exempla7, rycell-target m oleculing ofes the prese ntinvention in human- tumor bearin mice.g Human tumor cells that constituti velyexpre sslucifera andse display cell-surfa expressce ionof the targe oft the appropriate scFv-(n) are used in this xenograf model.t Methods known to the skilled worker are used to tes thet targe tedcytotoxicity7 of molecule ofs the present invention (see e.g. WO 2014/164680, WO 2014/164693, WO 2015/191764). Certa cell-tain rgeting molecules of the prese ntinvention are capable of significantly reducing the human tumor burden in mice challenge witd hhuman tumor cells. 872. 872. 872. id="p-872" id="p-872" id="p-872" id="p-872" id="p-872" id="p-872" id="p-872" id="p-872" id="p-872" id="p-872"
[872] As shown in Examples 1-4, combination Shiga toxin A Subunit effector polypepti desof the prese ntinvention may be used as scaffolds to create cell - targeting molecules that exhibit: 1) increas stability,ed 2) improved in vivo tolerability7 in chordate 2) s,reduc edimmunogenic potentia afterl administration to chordate and/os, ther ability to deliver an embedded or insert T-ceed ll epitope for MHC class I presentati by7on a nucleat chordated, cele l.In certai combinatn ions, the result ingleve ofl de-immunizatio repren sents tire synergistic action of individually de-immunized sub-regions which wer combie ned together.
Example 4. An Exemplary Cell-Targeti Moleng cule Target ingCD38+ CellsWO 2016/196344 PCT/US2016/034778 341 873. 873. 873. id="p-873" id="p-873" id="p-873" id="p-873" id="p-873" id="p-873" id="p-873" id="p-873" id="p-873" id="p-873"
[873] The binding character ofist theics "SLT-lA-combo7::aCD38-sc"Fv-l protein (SEQ ID NO:82) to an extracell humanular ,CD38 targe wast determined by a fluorescence-bas flow-cytomed, asetrysa y.Samples containi CD38ng positiv e (CD38+) cells (of either the cell line A or F) wer esuspended in IX PBS containing one percent bovine serum albumin (BSA) (Calbiochem, San Diego, CA, U.S.), hereinafte referrer to das "IX PBS+1%BSA", and incubated for one hour at 4°C wit h100 pL of vari ous dilutions of SLT-lA-combo7: aCD38-: scFv- l.The highes t concentra oftions SLT-lA-cornbo7:: aCD38-scFv-l teste ind the assay was select ed to sawrate all of the binding possibilit iesCD38. negative (CD38-) ceils (of tire cell lines H and I) were treated with the highest concentra oftion SLT-1A-combo?:: aCD38-scFv- testel ond targe positivt cellse in this assay. After the one hour incubation, the ceil samples were washe twicd withe IX PBS+1%BSA. The cel l samples wer incubae ted for one hour at 4°C with 100 uL of IX PBS+1%BSA containi a-SLT-lAng pAbl, then washed again and incubated for one hour at 4°C wit h100 pL of IX PBS+1%BSA solution containing an anti-rabbit secondar y antibody conjugated to fluoresce isointhiocya (FITC)nate. 874. 874. 874. id="p-874" id="p-874" id="p-874" id="p-874" id="p-874" id="p-874" id="p-874" id="p-874" id="p-874" id="p-874"
[874] The cell sampl eswer washede twic withe IX PBS+1%BSA, resuspended in 200 pL of IX PBS and subject edto fluorescence-bas flowed, cytometr to assy ay the percenta ofge cel lsbound by sufficie secondarynt 7 antibody, indicati ofve the binding level ofs SLT-lA-combo7: aCD38-: scFv- tol the cells in eac hsampl e.The data for all the samples in mean fluorescence intensity units (MFI), in relat ivefluorescence units, was obtaine byd gating the data using a negat ivecontr samol ple of cells which was not treated with any cell-targetin molegcule but which was incubated with the a-SLT-lA pAbl primary antibody and the anti-rabbit secondary antibody as describe above.d Tire integrated MFI (iMFI) was calculated by multiplying the percentage of positiv cellse with Ilie MFI. Graphs were plotted of iMFI vers us "concentra oftion protein" in nanomolar using Prism software (GraphPad Software , San Diego, CA, U.S.). Using the Prism software functi onof one-site bindin g[Y = Bmax*X / (Kd + X)] under the heading binding-saturation, the Bmax and Ku wer e calculat usinged baseli necorrected data. Bmax is the maximum specif icbinding reported in iMFI. Kd is the equilibrium binding constant, reported in nanomolar. 875. 875. 875. id="p-875" id="p-875" id="p-875" id="p-875" id="p-875" id="p-875" id="p-875" id="p-875" id="p-875" id="p-875"
[875] The Bmax for SLT-lA-combo7: aCD38-: scFv- (SEQl ID NO:82) binding to two, different, CD38+ cell types was measured to be approximatel 100,000y iMFI, and the Kd of SLT-lA-combo7: aCD38-: scFv- bindingl those CD38+ cel lswas WO 2016/196344 PCT/US2016/034778 342 measured to be approxima tely2--7 nM (Tabl e19; Figure 18). SLT-1 A-combo7:: aCD38-scFv- didl not exhibit specif icbinding to CD38- cells or high affinity binding to CD38- cells in this assa undery the conditions described (Figure 18).
Table 19. Binding Characteris Reprtics:esentat Valueive fors Bmax and Kd for Cell Binding by an Exempla ry,Cell-Target Molingecul ofe the Present Invention target positiv e targe positivet cell line A cell line F Kd Bmax Kd Bmax Cell-Target Moleing cule (iMFI) (nM) (iMFI) (nM) SLT-1 A-combo7:: 0tCD38-scFv-l 112,660 7.1 93,966 1.6 876. 876. 876. id="p-876" id="p-876" id="p-876" id="p-876" id="p-876" id="p-876" id="p-876" id="p-876" id="p-876" id="p-876"
[876] lire exempla ry,CL)38-target cell-ed, target moleingcule SLT-lA-combo7: : aCD38-scFv- wasl teste ford catalyti actcivity7 as describe ind Example 2. SLT-1A- combo?:: aCD38-scFv- (SEQl ID NO:82) exhibite ribosomed inactivati actonivit y comparable to a wild-type. 877. 877. 877. id="p-877" id="p-877" id="p-877" id="p-877" id="p-877" id="p-877" id="p-877" id="p-877" id="p-877" id="p-877"
[877] The exempla ry,CD38-targeted, cell-target moleingcule SLT-lA-combo7: : aCD38-scFv- wasl test edfor cytotoxic asity described in Example 2. SLT-1A- combo?:: aCD38-scFv-l (SEQ ID NO:82) was potentl cytoy toxic to CD38+ cells. 878. 878. 878. id="p-878" id="p-878" id="p-878" id="p-878" id="p-878" id="p-878" id="p-878" id="p-878" id="p-878" id="p-878"
[878] Tire exempla ry,CL)38-target cell-ed, target moleingcule SLT-lA-combo7: : «CD38-scFv- wasl teste ford reduced immunogenic7 asity describ edin Example 2.
SLT-lA-combo7: aCD38-s: cFv-l (SEQ ID NO:82) exhibited reduced immunogenicit as comparedy to a contr SLT-1A-ol FR:: aCD38-scFv-l. 879. 879. 879. id="p-879" id="p-879" id="p-879" id="p-879" id="p-879" id="p-879" id="p-879" id="p-879" id="p-879" id="p-879"
[879] The exempla ry,CD38-targeted, cell-target moleingcule SLT-1 A-combo7:: aCD38-scFv- wasl test edin a xenograf modelt of human cance usingr human cel ls expressing CD38 and an assa knowny to the skilled worker (see e.g. WO 2014/164693). SLT-lA-combo7: aCD38-sc: Fv-l (SEQ IL) NO:82) exhibited the ability to reduce tumor burden in mice injected with CD38 positive, human, neoplas cellstic as compar edto a control, vehicl treatee group.d In this study, mice wer einjected with 2.5 x 10° Daudi-Luc cel ls(a human, CD38 positiv tumore cell line that has been engineer toed expre ssthe luciferase gene ).Four days afte tumorr injection, mice were randomiz intoed groups of eight mice per group and treatmen t was started. The mice in the contr andol cell-target moleingcule treatme groupsnt received vehicle only or cell-targeti molecng ule, respectivel for y,twelve doses over five weeks (three times per week for two weeks, a wee kof no dosing then three times a week again for 2 weeks ).On differ entdays of the study whole, body bioluminescen (BLI)ce was measured in photons per second to monitor the tumorWO 2016/196344 PCT/US2016/034778 343 burden over time by detectin lucifeg rase express ingDaudi cell s.Before treatm ent, tiie mice wer erandomiz usinged their BLI readings in order to create mice groups with simila meanr and median BLI value s.The results of this study are reported in Table 20 and Figur e19. Table 20 lists the median BLI signal for the cel !-targeting molecule treate groupd and the vehicle-only control group and the percent of ־‘treated over control־’ (% T/C), defined by the formula: (media BLIn signa ofl the treated group) / (media BLIn signal of the vehicle-on contrly groupol x) 100. Day 4 involv pre-ed treatment measurements.
Table 20. Treatment with the Exempla ry,Cell-Target Moleculeing SLT-1A- _______ combo7::aCD38-scFv- Reducedl Tumor Burdens & Vivo_______ Treatment Group cell-targetin g SLT-1A-combo7:: aCD38- molecule vehicle-only scFv-1 BLI Percenta ofge tiie cell- Day of median BLI signal targeting molecule treated Study (photons / second) over control treated (% T/C) 94% 4 1.37e6 1.4666 7 1.6466 2.10e6 78% 14 7.66e6 9.3667 8% 22 4.22e9 9% 3.6068 28 1.8869 9.3269 20% 1.24610 3.26610 38% 40 1.65610 1.55611 11% 880. 880. 880. id="p-880" id="p-880" id="p-880" id="p-880" id="p-880" id="p-880" id="p-880" id="p-880" id="p-880" id="p-880"
[880] BLI measurements take onn study Days 7, 14, 22, 36, and 40 showed that the control vehic, le-only group had a highe tumorr burden than the group treate withd 0.5 milligra ofms SLT-lA-combo7: aCD38-: scFv- perl kg of body weight per dose.
At the fina time-pointl (Day 40), the percentage of BLI for the treated over the vehicle-only control (T/C) was 11%, indicating that there was an 89% reduct ionof tiie media tumorn burde nin the group treated wit hSLT-lA-combo7: aCD38-sc: Fv- I (SEQ ID NO :82).WO 2016/196344 344 Example 5. Cell-Targe Moltingecul Comprises ing a Shiga Toxin Effector Polypepti ofde the Present Invention Linked to a HERZ Binding Region («HER2-VhH fused wit hSLT-lA-combo(n)) 881. 881. 881. id="p-881" id="p-881" id="p-881" id="p-881" id="p-881" id="p-881" id="p-881" id="p-881" id="p-881" id="p-881"
[881] In this example, any one of the Shiga, toxin effector polypepti desSLT-1 A- comboO-26, as describe above,d is operabl linkedy to an immunoglobul bindingin region anti-HER2, which binds specifica andlly wit hhigh-affinity to an extracellula r antigen on huma nHER2, such as the single-domain variable region of the camdid. antibody 5F7 (0HER2-VhH) as described in U.S. Patent Application Publication 2011/59,090, to form an exemplary, cell-targeting molecule of the prese ntinvention.
Construction, Production, and Purification of the Cytotoxic, HER2-Binding. Fusion Protein"SLT-1A-combon):aHER2-V„H" 882. 882. 882. id="p-882" id="p-882" id="p-882" id="p-882" id="p-882" id="p-882" id="p-882" id="p-882" id="p-882" id="p-882"
[882] For certai embodimen nts, the immunoglobulin-derive bindingd regio n 0HER2-VhH and Shiga toxin effector polypept ideare fused together to form, a single, continuous polypept ide"SLT-1 A-combo(n)::aHER2-VHH." In this example, a polynucleot encideoding oHER2-VhH, a bindin gregion comprisin a gVhH variable domai derin ved from the immunoglobul 5F7,in is clone ind fram wite ha polynucleotid encodinge a linker known in the art and in frame with a polynucleotid encodinge the Shiga toxin effector polypeptide SLT-lA-combo(n).
Expression of the cell-targeting molecule SLT-lA-combo(n)::aHER2-VHH is accomplis hedusing either bacter ialand/o cell-fr ree, protein translat systemion s known to the skill edworker and/o asr describ edin the previous examples.
Determining the In Vitro Characteristics of SLT-lA-combo(n)::aHER2-VHH 883. 883. 883. id="p-883" id="p-883" id="p-883" id="p-883" id="p-883" id="p-883" id="p-883" id="p-883" id="p-883" id="p-883"
[883] The binding character ofist theics cell-targetin molegcule of this example for HER2+ cells and HER2- cells is determined by a fluorescence-bas flowed, - cytometr They. Bmax for SLT-lA-combo(n)::aHER2-VHH to HER2+ cel lsis measured to ,be approximatel 50,000-2y 00,000 MFI with a Kd within the range of 0.01-100 nM, wherea theres is no significa bindinnt tog HER2- cells in this assa y. 884. 884. 884. id="p-884" id="p-884" id="p-884" id="p-884" id="p-884" id="p-884" id="p-884" id="p-884" id="p-884" id="p-884"
[884] The ribosome inactivati abilitieson of the SLT-lA-combo(n)::aHER2-VHH cell-target moleculeing is determined in a cell-fr inee, vitro protei transn lation as described above in the previous example Thes. inhibitory effect of the cell-targetin g molecule of this exampl one cell-free protei synthesisn is significant. The IC50 ofWO 2016/196344 PCT/US2016/034778 345 SLT-lA-combo(n)::aHER2-VH onH protei synthesisn in this cell-fre asesa isy approxima tely0.1-100 pM.
Determining the Cytotoxici ofty SLT-1 A-combo(n)::aHER2-VHH Using a HER2+ Cell-Kill Assay 885. 885. 885. id="p-885" id="p-885" id="p-885" id="p-885" id="p-885" id="p-885" id="p-885" id="p-885" id="p-885" id="p-885"
[885] The cytotoxici characty teristics of SLT-1A-combo(n):aHER2-VHH is determined by the genera cell l-ki assll ay as describe aboved in the previous examples using HER2+ cells. In addition, the selective cytotoxicity characterist ics of SLT-lA-combo(n)::aHER2-V HHis determined by the same general cell-kill assay using HER2- cells as a comparison to the HER2+ cell s.The CD50 of the cell- targeting molecule of this example is approximately 0.01-100 nM for HER2+ cel ls depending on the ceil line. Hie CD50 of SLT-lA-combo(n)::aHER2-VHH is approximat 10-10,000ely fold great (lesser cytotoxic) for cells not expressing HER2 on a cellular surfac ase compared to cells which do express HER2 on a cellular surface. In addition, the cytotoxici ofty' SLT-lA-combo(n)::aHER2-VHH is investigate for bothd dire ctcytotoxici andty indirect cytotoxic byity T-cell epitop e delivery' and presentation leading to CTL-mediated cytotoxici usingty assa ysknown to the skilled worker and/o descr ribe herein.d Determin ingthe// ?Vivo Effects of the Exempla ry.Cell-Targeting Molecul SLT-e lA-combo(n)::aHER2-VnH Using Anima Modelsl 886. 886. 886. id="p-886" id="p-886" id="p-886" id="p-886" id="p-886" id="p-886" id="p-886" id="p-886" id="p-886" id="p-886"
[886] Anima modelsl are used to determine the in vivo effects of the cell-targetin g molecule SLT-lA-combo(n)::aHER2-VHH toward neoplas cellstic Vario. us mice strai arens used to tes thet effec oft SLT-1 A-combo(n)::aHER2-VnH after intravenous administrati on onxenogr tumorsaft in mice result ingfrom the injection into those mice of human neoplas cellstic whic hexpress HER2 on their cei l surfaces Cell. killing is investigate for bothd direc cytott oxici andty indire ct cytotoxic byity T-cell epitope delivei andy presenta tionleading to CTL-mediated cytotoxici usingty assays known to the skill edworker and/o descr ribe herein.d 30WO 2016/196344 PCT/US2016/034778 346 Example 6. Cell-Targeti Moleculng Compries sing a Shiga Toxin A Subunit Effect Polypeor pti ofde the Present Invention, a Fused, T-Cel lEpitope-Pepti de, and a ligand Binding Region Specifi toc IL-2R 887. 887. 887. id="p-887" id="p-887" id="p-887" id="p-887" id="p-887" id="p-887" id="p-887" id="p-887" id="p-887" id="p-887"
[887] In this example, the Shiga toxin effector polypeptide is derive fromd the A subunit of Shiga-like Toxin 1 (SLT-1A) as describe above,d optionall withy amino aci dresidue substitutions conferr funn-ing cleav resageistance such, as, e.g., R248A/R251A (see Exampl 3,e supra). A human, CD8+ T-cell epitope-pept iside selected based on MHCI molecule bindin predicg tions, HLA type s,already characteriz immunogeniced itie ands, readily availa blereagents as described above, such as the epitope GVMTRGRLK (SEQ ID NO:560). A binding regio isn derived from a ligand (the cytoki interne leukin 2 or IL-2) for the human interleukin 2 receptor (IL-2R), which is capable of specifically binding an extracellular part of tire human IL-2R. IL-2R is a cell-surfa recceeptor expressed by vario usimmune cell types such, as T-cells and natural killer cells.
Construction, Production, and Purification of the Cell-Target Fusioning Proteins T- epitope:: SET-1A:: IL-2 and IL-2::T-epitop:: eSLT-1A 888. 888. 888. id="p-888" id="p-888" id="p-888" id="p-888" id="p-888" id="p-888" id="p-888" id="p-888" id="p-888" id="p-888"
[888] The ligand-type binding regio aIL-2R,n the Shiga, toxin effector polypeptide, and T-cell epitope are linked togeth toer form a single continu, ous polypepti de,such as "T-epitope::SLT-lA::lL-2" or ‘IL-2: :T-epitope:: SLT-1 A," and, optionally, a KDEL is added to the carboxy-termi ofnus the result ingpolypeptide For. example, fusion proteins are produced by express ingfrom polynucleoti encodingdes T- epitope::SLT-lA::IL-2 and IL-2 ::T-epitope ::SLT-LA. Expression of the T- epitope::SLT-1 A::IL-2 or IL-2::T-epitope::SLT-1A cell-target molecing ules is accomplished using either bacterial and/o cell-fr ree, protein translation systems as describe ind the previous examples.
Determin ingthe In Vitro Characteri ofsti thecs Cell-Target Molecing ules SLT- 1A: :T-epitop e::IL-2 and IL-2::SLT-1 A::T-epitope 889. 889. 889. id="p-889" id="p-889" id="p-889" id="p-889" id="p-889" id="p-889" id="p-889" id="p-889" id="p-889" id="p-889"
[889] The binding character ofist cell-ics target moleculesing of this example for IL-2R positive cells and IL-2R negativ cellse is determined by fluorescence-bas ed, flow-cytome Thetry. Bmax for both T-epitope::SLT-lA::IL and-2 IL-2::T- epitope:: SLT-1A to IL-2R positive cells is measure tod be approximat 50,000-ely WO 2016/196344 PCT/US2016/034778 347 200,000 MFI with a Kd withi then range of 0.01-100 nM, where thereas is no significa bindingnt to IL-2R negat ivecells in this assay. 890. 890. 890. id="p-890" id="p-890" id="p-890" id="p-890" id="p-890" id="p-890" id="p-890" id="p-890" id="p-890" id="p-890"
[890] The ribosome inactivati abilonitie ofs T-epitope ::SLT-1 A::IL-2 and IL-2::T- epitope: :SLT-1 A are determined in a cell-fr inee, vitro prote transin lation as describe aboved in the previous example Thes. inhibitory effect of the ceil-targetin g molecules of this exampl one cell-free protein synthesis is significant. For both T- epitope::SLT-lA::IL-2 and IL-2 ::T-epitope:: SLT-1 A, the IC50 of on protein synthes inis this cell-fre asse ay is approximat 0.1-100ely pM.
Determin ingthe Cytotoxici ofty the Cell-Target Molecing ules T-epitope ::SLT- TA::IL-2 or IL-2::T-epitope::SLT-1 A Using a Cell-Kil Assal y 891. 891. 891. id="p-891" id="p-891" id="p-891" id="p-891" id="p-891" id="p-891" id="p-891" id="p-891" id="p-891" id="p-891"
[891] The cytotoxic charity acter ofist T-epitics ope::SLT-lA::IL- or2 IL-2::T- epitope::SLT-1 A are determined by the genera cell-l kill assay as described above in the previous examples using IL-2R positive cells In. addition, the selecti ve cytotoxic chaityracteris of ticsT-epitope:: SLT-1A: :IL-2 or IL-2::T-epitope:: SLT-1A are determined by the same general ceil-kill assa usingy IL-2R negative ceils as a comparison to the IL-2R positive cell s.The CD50 of the cell-targeti molecng ules of this example is approximat 0.01-10ely 0 nM for IL-2R positive cel lsdepending on the cell line. The CD50 of T-epitope::SLT-lA::IL- or2 IL-2::T-epitope::SLT-1 A is approximat 10-10,000ely fold great (lesser cytotoxic) for cells not express ingIL-2R on a cellular surfac ase compared to cells which do express IL-2R on a cellular surface. In addition, the cytotoxici of T-epitty ope::SLT- A::IL-1 2 or IL-2: :T- epitope::SLT-1 A is investigate for bothd direc cytotoxicityt and indirect cytotoxic ity by T-cell epitope deliver}׳ and presenta tionleading to CTL-mediated cytotoxici ty using assa ysknown to the skille workerd and/o descrr ibed herein.
Determin ingthe//? Vivo Effects of the Cell-Target Moleculeing T-epits ope: :SLT- 1A::IL2־ or IL-2: :T-epitope: :SLT-1A. Using Animal Models 892. 892. 892. id="p-892" id="p-892" id="p-892" id="p-892" id="p-892" id="p-892" id="p-892" id="p-892" id="p-892" id="p-892"
[892] Anima modelsl are used to determine the in vivo effects of the cell-targetin g molecules T-epitope::SLT-lA::IL- and2 IL-2::T-epitope::SLT-1 A on neoplast ic cells. Various mice strain ares used to tes thet effect of T-epitope::SLT- 1A::IL-2 and IL-2::T-epitope::SLT-lA after intravenous administration on xenograf tumorst in mice result ingfrom the injecti oninto those mice of human neoplas cellstic which express IL-2R on their cell surfac es.Cell killing is investigate for bothd dire ctWO 2016/196344 PCT/US2016/034778 348 cyiotoxic andity indirec cytotoxict byity T-celi epitope delivery and presentati on leading to CTL-mediated cytotoxicity using assa ysknown to the skilled worker and/o descrr ibed herein.
Example 7. Cell-Target Molecing ules Comprising a Shiga Toxin Effector Polypepti ofde the Prese ntInvention Linked to a CEA Bindin Regig on (aCEA- (Fn3) binding region fused wit hSLT-lA-combo(n)) 893. 893. 893. id="p-893" id="p-893" id="p-893" id="p-893" id="p-893" id="p-893" id="p-893" id="p-893" id="p-893" id="p-893"
[893] In this example, the Shiga toxin effector polypeptide SLT-lA-combo(n), as describe above,d is operably linked to an immunoglobulm-type, bindin gregion anti- CEA (Fn3) binding region, which binds specifica andlly with high-affinity' to an extracellular antigen on human carcinoembryonic antigen (CEA), such as the tenth human fibronec typetin III domain derived bindin regg ion C743 as describe ind Piri e C et al., J Biol Chem 286: 4165-72 (2011), to form an exempla ry,cell-targetin g molecule of the present invention. In addition, an immunogenic, CD8+ T-cell epitope is fuse dto the amino-term inusof the Shiga toxin effector polypeptide of this example to fonn epitope-SLT-lA-combo(n).
Construction, Production, and Purification of the Cytotoxic CEA-B. ؛nding. Fusion Protein "aCEA-(Fn3) fused with epitope-SLT-1 A-combo(n" ) 894. 894. 894. id="p-894" id="p-894" id="p-894" id="p-894" id="p-894" id="p-894" id="p-894" id="p-894" id="p-894" id="p-894"
[894] For certain embodiments, the immunoglobulin-type bindin gregion aCEA- (Fn3) and Shiga toxin effector polypeptide are fused together to fonn a singl e, continuous polypept ide"aCEA-(Fn3) fuse dwith epitope-SLT-1 A-combo(n" ).In this example, a fusion protein ،،aCEA-(Fn3) fused with epitope-SLT-lA-combo(n)״ is designe andd produced as describe ind the previous examples.
Determining the//? Vitro Characteri ofsti ،،aCEA-(cs Fn3) fused wit hepitope-SLT- lA-combo(n)" 895. 895. 895. id="p-895" id="p-895" id="p-895" id="p-895" id="p-895" id="p-895" id="p-895" id="p-895" id="p-895" id="p-895"
[895] The binding character ofist theics cell-targetin molegcule of this example for CEA+ cells and CEA- cells is determined by a fluorescence-bas flow-cytometred, y.
The Bmax for aCEA-(Fn3) fused with epitope-SLT-lA-combo(n) to CEA+ cells is measured to be approxima tely50,000-200,000 MFI with a Ku within the range of 0.01-100 nM, where theras ise no significa bindinnt tog CEA- cells in this assay. 896. 896. 896. id="p-896" id="p-896" id="p-896" id="p-896" id="p-896" id="p-896" id="p-896" id="p-896" id="p-896" id="p-896"
[896] The ribosome inactivati abilonitie ofs the aCEA-(Fn3) fused with epitope - SLT-lA-combo( cen) ll-targeti molengcule is determined in a cell-free, in vitroWO 2016/196344 PCT/US2016/034778 349 protei transn lati as ondescribe aboved in the previous example Tires. inhibitory effect of the cell-targeti molecng ule of this exampl one cell-fre proteie synthesisn is significant The. IC50 of aCEA-(Fn3) fused with epitope-SLT-1 A-combo(n on) protein synthesis in this cell-fre asesa isy approximat 0.1-100ely pM.
Determin ingthe Cytotoxicity7 0P‘aCEA-(Fn3) fuse dwith epitope-SLT-1 A- combo(n)" Using a CEA+ Cell-Kil Assl ay 897. 897. 897. id="p-897" id="p-897" id="p-897" id="p-897" id="p-897" id="p-897" id="p-897" id="p-897" id="p-897" id="p-897"
[897] The cytotoxic characity teristics of aCEA-(Fn3) fused with epitope-SLT A--1 combo(n) is determine by dthe general cell-kill assay as describe aboved in the previous examples using CEA+ cells In. addition, the selective cytotoxici ty' character ofist aCEA-(ics Fn3) fuse dwith epitope-SLT-lA-comb iso(n) determin ed by the same gener cell-al kill assa usingy CEA- cells as a comparison to the CEA+ cells. The CD50 of the cell-target molecing ule of this example is approxima tely 0.01-100 nM for CEA+ cells dependi ngon the cell line. The CD50 of aCEA-(Fn3) fused wit hepitope-SLT-1 A-combo(n) is approxima tely10-10,000 fold great (leser s cytotoxic) for cells not express ingCEA on a cellular surface as compared to cel ls which do expre ssCEA on a cellular surface. In addition, the cytotoxici7 ofty aCEA- (Fn3) fuse dwith epitope-SLT-lA-comb iso(n) investigate for bothd dire ct cytotoxic andity indirec cytotoxict byity T-cell epitope delivery7 and presentati on leading to CTL-mediated cytotoxic usingity assa ysknown to the skilled worker and/or described herein.
Determining theF/vo Effects of the Exempla ry,Cell-Targeting Molecul "aCEA-e (Fn3) fuse dwith epitope-SLT-1 A-combo(n" )Using Animal Models 898. 898. 898. id="p-898" id="p-898" id="p-898" id="p-898" id="p-898" id="p-898" id="p-898" id="p-898" id="p-898" id="p-898"
[898] Animal models are used to determine the in vivo effects of the cell-targetin g molecule aCEA-(Fn3) fused with epitope-SLT-lA-comb towardo(n) neoplast ic cells. Various mice strain ares used to tes thet effect of aCEA-(Fn3) fused wit h epitope-SLT-1 A-combo(n) after intravenous administration on xenograf tumorst in mice resul ting from the injecti oninto those mice of human neoplas cellstic which express CEA on thei cellr surfac es.Cell killing is investigate for bothd dire ct cytotoxici andty indirec cytotoxict byity T-cell epitope delive andry presentat ion leading to CTL-mediated cytotoxic usingity assa ysknown to the skilled worker and/o descrr ibed herein.Example 8. Cytotoxic, cell-targeting molecules comprising a Shiga toxin effector polypeptide SLT-lA-combo(n) and the antibody ahelminth-intestinal-antigen 899. 899. 899. id="p-899" id="p-899" id="p-899" id="p-899" id="p-899" id="p-899" id="p-899" id="p-899" id="p-899" id="p-899"
[899] In this example, any one of the Shig atoxin effector polypeptides SLT-lA-comboO- 26, as described above, is operationall linkedy to an immunoglobulin-t bindingype region target aing helminth antigen. An immunoglobulin-type binding region ahelminth-intesti nal- antigen is derived from an antibody generated, using technique knowns in the art, to the helminth ortho logof a human transferri receptn (seeor e.g. the nematode gene gcp-2.1 UniProt G8JYE4_CAEEL; Rosa B et al., Mol Cell Proteomics Ml 14.046227 (2015)).
Constructi Producton, ion, and Purificatio of nthe Cytotoxic, Cell-Targeti Moleng cul "SET-e 1 A-combo(n):: aHelminth-Intestinal"-Antigen 900. 900. 900. id="p-900" id="p-900" id="p-900" id="p-900" id="p-900" id="p-900" id="p-900" id="p-900" id="p-900" id="p-900"
[900] The immunoglobulin-type binding region ahelminth-intestinal-an and Shigatigen toxin effector region, which is optional a proteasly e-cleavage resistant Shiga toxin effect or region are, linked together to form a protei inn which the immunoglobulin-type binding region is not locat proximaed to lthe protein’s amino-terminus as compared to the Shiga toxin effector region For. example, a fusion protei isn produced by express inga polynucleotide encoding an ahelminth-intestinal-antigen-binding prote fusedin to an amino-termina SET-l lA-comboO-26. Expressi onof the SLT-lA-combo(n)::ahelminth-intestinal-anti gen-binding protei isn accomplishe usingd either bacteri and/oral cell-free, protein translation systems as described in the previous examples.
Determin ingthe In Vitro Character istof theics Cytotoxi Cellc, -Targeti Moleng cul SLT-1e A- combo(n): :aHelminth-Intestinal-Antigen 901. 901. 901. id="p-901" id="p-901" id="p-901" id="p-901" id="p-901" id="p-901" id="p-901" id="p-901" id="p-901" id="p-901"
[901] The binding characteri ofstic the cytots oxic, cell-targeti molngecule of this exampl ise determined by a molecul bindingar assay known in the art using a purified, recombinant, target protei n.The Kpfor SLT-1 A: :ahelminth-intestinal and/or-anti SLT-1gen A- FR::ahelminth-intestinal- to antitarget genprotei isn measured to be approximatel 100 ynM, whereas there is no significant binding to a negat ivecontrol prote (e.g.in purified, recombinant, helminth orthol ofog human transferrin receptor) in this assay. 350WO 2016/196344 351 902. 902. 902. id="p-902" id="p-902" id="p-902" id="p-902" id="p-902" id="p-902" id="p-902" id="p-902" id="p-902" id="p-902"
[902] The ribosome inactivati abilion ties of the SLT-1A: :ahelminth-intes tinal- antigen is determine in ad cell-free in vitro, protein translation as described above in the previous example Thes. inhibitor effy ect of the cytotoxic, cell-targetin g molecule of this example on cell-free protei synthesisn is significant. Tire IC50 of SLT-1 A: :ahelminth-intestinal-ant on proteiigen syntn hes inis this cell-f reeassay is approximat 0.1-100ely pM.
Determining the Toxicit ofy the Cytotoxic Cell-, Target Moleing cule SLT-1 A- combo(n)naHelminth-Intestinal-Antigen 903. 903. 903. id="p-903" id="p-903" id="p-903" id="p-903" id="p-903" id="p-903" id="p-903" id="p-903" id="p-903" id="p-903"
[903] The toxicity of SLT-1 A::ahelminth-intestinal- to antigenhelminths is determined using model helminths (see e.g. latsenko I et al., Toxins 2050-63 (2014)). The helminth can be administered purified SLT-1A:: ahelminth-intes tinal- antigen by soaking or alternatively by feeding the helminth with bacteria expressing the SLT-1 A::ahelminth-intestinal- fusiantigeon protein n. 904. 904. 904. id="p-904" id="p-904" id="p-904" id="p-904" id="p-904" id="p-904" id="p-904" id="p-904" id="p-904" id="p-904"
[904] In addition, laboratory' animals harbori helming nths and/o displayingr helminth-rela diseaseted ares administered SLT-lA::ahelminth-intestinal- antigen and monitor fored reduction or eliminat ofion helminths and/or associated symptoms of parasitic helmint (s)h.
Example 9. Cell-target moleingcules comprising a Shig atoxin effector polypepti SLT-lAde -combo(n) fused to an immunoglobulin-type binding region specific to HIV-1 Gag 905. 905. 905. id="p-905" id="p-905" id="p-905" id="p-905" id="p-905" id="p-905" id="p-905" id="p-905" id="p-905" id="p-905"
[905] In this example, any one of the Shiga toxin effector polypept ideSLT-1 A- comboO-26, as describe above,d is operably linked to an immunoglobulm-type binding region aGag-antige whichn, is derived from an immunoglobulin-ty pe domain recognizi theng HIV capsid protein HIV-1 Gag polyprote (seein e.g. Nagola S et al., Retrovirology 9: 17 (2012)) and which compris anes artifici Ankyrinal, domai repeatn polypeptide capable of binding an extracellular part of Gag. Gag is the major structural protei involven ind HIV vims assembly and oligomer izeintos a. lattic ofe as many as 700 to 5000 copie pers virion to form a conical-shaped CA core (see e.g. Chen Y et al., Biophys J 96: 1961-9 (2009); Pomillos O et al., Nature 469: 424-7 (2011)).WO 2016/196344 PCT/US2016/034778 352 Construction, Production, and Purification of the Cell-Target Molecing ules "SLT- lA-combo(n) linked to aGag" 906. 906. 906. id="p-906" id="p-906" id="p-906" id="p-906" id="p-906" id="p-906" id="p-906" id="p-906" id="p-906" id="p-906"
[906] The immunoglobulin-t bindinype gregio aGagn and Shiga toxin effector polypept ideSLT-lA-combo(n) are fused to form a. cytotoxic protein. For example , a fusion protei isn produced by expressing a polynucleot encodingide the aGag- antigen-bindi proteinng SLT-lA-combo(n)::aGag Expre. ssion of the SLT-1A- combo(n)::aGag cytotoxic protei isn accomplis hedusing either bacter and/orial cell- free protein, translation systems as describe ind the previous examples.
Determining the Jr Vitro Character isticof the sCell-Targeting Molecul "SLT-1e A- combo(n)::aG"ag 907. 907. 907. id="p-907" id="p-907" id="p-907" id="p-907" id="p-907" id="p-907" id="p-907" id="p-907" id="p-907" id="p-907"
[907] The binding character ofist theics cytotoxic protei ofn this example for purified, recombinant HIV-1 Gag is determin byed an assay known in the art, such as an enzyme-linke immunosorbentd assay. The Kd for SLT-lA-combo(n)::aG ag binding to Gag is measured to be approxima telywithin the range of 0.01-100 nanomolar (nM). 908. 908. 908. id="p-908" id="p-908" id="p-908" id="p-908" id="p-908" id="p-908" id="p-908" id="p-908" id="p-908" id="p-908"
[908] The ribosome inactivati abilitieson of the SLT-1 A-combo(n)::aG cytotoxicag protein is determined in a cell-free, in vitro protein translation as described above in the previous example Thes. inhibitory7 effec oft the cytotoxic protein of this example on cell-f reeprotein synthesis is significant. The IC50 of SLT-lA-combo(n)::aGa g on protei synthesisn in this cell-f reeassay is approximatel 0.1-100y pM.
Determining the Cytotoxici ofty the Cell-Target Moleculeing "SLT-1A- comboin)::aGa" Usingg a Cell-Kill Assay 909. 909. 909. id="p-909" id="p-909" id="p-909" id="p-909" id="p-909" id="p-909" id="p-909" id="p-909" id="p-909" id="p-909"
[909] The cytotoxic characity teristics of SLT-1 A-combo(n)::aGag are determine d by the general ceil-kill assa asy describe aboved in the previous examples using HIV-infecte humand T-cells. In addition, the selective cytotoxicity character ofist ics SLT-lA-combo(n)::aG areag determine by dthe same general cell-kill assay using uninfected T-cells as a comparison to the infected T-cells The. CD50 of the cytotoxic protein of this example is approximatel 0.01-100y nM for infecte T-celd ls wit hactively replicating virus. The CD50 of the cytotoxic protein is approxima tely -10,000 fold great (lesser cytotoxic) for uninfected T-cells.Determining the In Vivo Effects of the Cytotoxic, Cell-Target Moleing cul "SLT-1e A- combo(n)::aG"ag Using Animal Models 910. 910. 910. id="p-910" id="p-910" id="p-910" id="p-910" id="p-910" id="p-910" id="p-910" id="p-910" id="p-910" id="p-910"
[910] The use of SLT-1 A-combo(n)::aGag to inhib itthe progress ofion HIV infection is tested by administering SLT-1 A-combo(n)::aGag to simian immunodeficiency virus (SIV) infect ednon-human primat (seees Selli erP et al,, PL0S One 5: 610570 (2010)).
Example 10. A cytotoxic, cell-targeting molecule derived from the A Subunit of Shiga toxin and the antibody ahistoplasma-antigen 911. 911. 911. id="p-911" id="p-911" id="p-911" id="p-911" id="p-911" id="p-911" id="p-911" id="p-911" id="p-911" id="p-911"
[911] In this example, any one of the Shiga toxi effectn polypeor ptide SLT-1s A-comboO - 26, as described above, is operably linked to an immunoglobulin-type binding region ahistoplasma-ant whichigen, is derived from a known antibody or an antibody generated, using technique knowns in the art, to a Histoplasma capsulatum surface antigen (see e.g., H. capsulatum H antigen (Deepe G, Durose G, Infect Immun 63: 3151-7 (1995)) and the mAb H1C (Lopes L et al., Clin Vaccine Immunol 17: 1155-8 (2010); H. capsulatum, cel surfacl e, histone-l proteiike H2Bn (Nosanchuk J et al., J Clin Invest 112: 1164-1175 (2003))).
Construct Produion, cti on,and Purificat ionof the Cytotoxic Cell-Target Moleculing SLT-e 1 A-combo(n): :aHistoplasma-Antigen 912. 912. 912. id="p-912" id="p-912" id="p-912" id="p-912" id="p-912" id="p-912" id="p-912" id="p-912" id="p-912" id="p-912"
[912] The immunoglobulin-typ bindinge region ahelminth-intestinal- andanti Shigagen toxi effen ctor polypeptide which, is optiona ally protease-cleavage resista Shigant toxi n effect region,or are linked togethe to formr a prote inin which the immunoglobulin-t ype binding region is not located proxim toal the protein’s amino-terminus as compared to the Shiga toxi effen ctor polypepti de.For example, a fusion prote isin produced by express inga polynucleoti encodingde a Histoplasma-surface-antigen prote-bin dingfusedin to an amino­ terminal, SLT-1 A-combo(n). Expression of the SLT-1 A-combo(n)::aHistoplasma-anti gen- binding protei isn accomplis hedusing either bacterial and/or cell-free, prote translatin ion systems as described in the previous examples. 353WO 2016/196344 PCT/US2016/034778 354 Determining the In Vitro Character isticof the sCytotoxic, Cell-Target Moleculeing SLT-lA-combo(n)::aHistoplasma-Antigen 913. 913. 913. id="p-913" id="p-913" id="p-913" id="p-913" id="p-913" id="p-913" id="p-913" id="p-913" id="p-913" id="p-913"
[913] The binding character ofist theics cytotoxic, cell-target moleingcule of this example is determined by a molecul bindinar asg sa knowny in the art using a purified recombina targent protein.t The Kd for SLT-lA-combo(n)::aHistoplasma - antigen to targe proteint (e.g. a purified, recombinant, H. caps!,datum surfa ce antige isn) measured to be approxima tely100 nM, wherea theres is no significant binding to a negativ contre proteiol inn this assay. 914. 914. 914. id="p-914" id="p-914" id="p-914" id="p-914" id="p-914" id="p-914" id="p-914" id="p-914" id="p-914" id="p-914"
[914] Ilie ribosome inactivati abilitieson of the SLT-lA-combo(n)::aHistoplas ma- antigen cytotoxic prote inis determined in a cell-free in vitr, o protei transn lation as describe aboved in the previous example Thes. inhibitory effect of the cytotoxic, cell-targetin molegcule of this exampl one cell-free protei synthesisn is significant.
The IC50 of SLT-1A-combo(n)aHistoplasma -anton proteinigen synthesis in this cell-fre asesa isy approximat 0.1-100ely pM.
Determining the Anti-Fungal Activity of the Cytotoxi Ceil-Tac, rgeting Molecule SLTMA-combo(n): :aHistoplasma-Ant Usingieen Fungi 915. 915. 915. id="p-915" id="p-915" id="p-915" id="p-915" id="p-915" id="p-915" id="p-915" id="p-915" id="p-915" id="p-915"
[915] The fungicida actl ivity of SLT-1 A-combo(n)::aHistopIasma-ant to igen funga ceilsl is determined. Purifie d,StxA::aHistoplasma-a and/ontigen StxA-r FR::aHistoplasma-anti is diregenctly administere to fungad culturl ines order to measure fungicidal activ ity(see e.g. Li R et al., Antimicrob Agents Chemother 44: 1734-6 (2000)). In addition, laboratory animals infecte witd hfungi (e.g. H. capsulatum) and/or displaying histoplasmos sysis,temic mycose and/ors, other FT c47psM/<2/M7׳«-related disease ares administer SLT-lA-ced ombo(n)::aHistopla sma- antigen and monitored for reduct ionor elimination of funga pathl ogens and/or associated symptoms of funga infel ctions (see e.g. Kobayashi G et al., Antimicrob Agents Chemother 34: 524-8 (1990)).
Example 11. Cytotoxic Cell-Targeti Molngecul Targetes ingVarious Cell Types 916. 916. 916. id="p-916" id="p-916" id="p-916" id="p-916" id="p-916" id="p-916" id="p-916" id="p-916" id="p-916" id="p-916"
[916] In this example, the Shiga toxin effector regio isn derived from the A subunit of Shiga-like Toxin 1 (SLT-1 A), Shiga toxin (StxA), and/or Shiga-like Toxin 2 (SLT-2A) such that it compris aes combination of sub-regions describe hered into provide two or more of the following: 1) de-immunization, 2) protease-cle avage resistanc and/oe, 3)r an embedded or inserte heted, rologous T-ce,ll epitope A. WO 2016/196344 PCT/US2016/034778 355 binding regio isn derived from the molecule choss en from colum n1 of Table 21 and which binds tire extracellula target rbiomolec uleindicated in colum 2n of Table 21.
The resulting combinatio Shigan, toxin effector polypepti desand binding regions are fused together to from various single-chain, polypeptides The. exemplar proteinsy of this example are optional creatly withed a carboxy-termina KDEL-tl ype signa l motif using techniques known in the art and optionally linked to an additional exogenou maters ial, such, as, a detect ionpromoti agent(ng s).The exemplary proteins of this exampl aree test edas describe ind the previous examples using cell s expressing the appropriate extracellu targlar biomoleet cule Thes. exemplary proteins of this exampl maye be used, e.g., to labeling subcellula compar rtme ofnts targe celt lsand to diagnose and treat diseases conditi, ons, and/or disorders indicated in column 3 of Table 21.
Table 21, Various Binding Regions for Cell-Targeti of ngCytotoxic Proteins Source of Extracellular binding region target Application(s) alemtuzumab CD52 B-cell cancers, such as lymphoma and leukemia, and B-cell related immune disorders, such as autoimmune disorders basiliximab CD25 T-cell disorders, such as prevention of organ transplant rejections, and some B-cell lineage cancers CD30 hematological cancers, B-cell related immune brentuximab disorders, and T-cell related immune disorders catumaxomab EpCAM various cancers, such as ovarian cancer, malignant ascites, gastric cancer cetuximab EGFR various cancers, such as colorectal cancer and head and neck cancer daclizumab CD25 B-cell lineage cancers and T-cell disorders, such as rejection of organ transplants daratumumab CD38 hematological cancers, B-cell related immune disorders, and T-cell related immune disorders ganglioside Various cancers, such as breast cancer, myeloid dinutuximab GD2 cancers, and neuroblastoma efalizumab LFA-1 autoimmune disorders, such as psoriasis (CD Ila) ertumaxomab HER2/neu various cancers and tumors, such as breast cancer and colorectal cancer gemtuzumab CD33 myeloid cancer or immune disorder CD20 B-cell cancers, such as lymphoma and leukemia, ibritumomab and B-cell related immune disorders, such as autoimmune disorders CD152 T-cell related disorders and various cancers, such ipilimumab as leukemia, melanoma muromonab CD3 prevention of organ transplant rejections natalizumab integrin a4 autoimmune disorders, such as multiple sclerosis and Crohn’s diseaseWO 2016/196344 PCT/US2016/034778 356 obinutuzumab CD20 B-cell cancers, such as lymphoma and leukemia, and B-cell related immune disorders, such as autoimmune disorders ocaratuzumab CD20 B-cell cancers, such as lymphoma and leukemia, and B-cell related immune disorders, such as autoimmune disorders CD20 B-cell cancers, such as lymphoma and leukemia, ocrelizumab and B-cell related immune disorders, such as autoimmune disorders ofatumumab CD20 B-cell cancers, such as lymphoma and leukemia, and B-cell related immune disorders, such as autoimmune disorders palivizumab F protein of treat respiratory syncytial virus respiratory syncytial vims panitumumab EGFR various cancers, such as colorectal cancer and head and neck cancer pertuzumab HER2/neu various cancers and tumors, such as breast cancer and colorectal cancer pro 140 CCR5 HIV infection and T-cell disorders ramucirumab VEGFR2 various cancers and cancer related disorders, such as solid tumors CD20 rituximab B-cell cancers, such as lymphoma and leukemia, and B-cell related immune disorders, such as autoimmune disorders tocilizumab or IL-6 receptor autoimmune disorders, such as rheumatoid atlizumab artliritis tositumomab CD20 B-cell cancers, such as lymphoma and leukemia, and B-cell related immune disorders, such as autoimmune disorders trastuzumab HER2/neu various cancers and tumors, such as breast cancer and colorectal cancer ublituximab CD20 B-cell cancers, such as lymphoma and leukemia, and B-cell related immune disorders, such as autoimmune disorders integrin a4p7 autoimmune disorders, such as Crohn’s disease vedolizumab and ulcerative colitis CD20 binding CD20 B-cell cancers, such as lymphoma and leukemia, antibodies and and B-cell related immune disorders, such as scFv(s) autoimmune disorders (see e.g. Geng S et al., Cel! Mol Immunol 3: 439-43 (2006); OlafesnT et al., Protein Eng Des Sei 23: 243-9 (2010)) CD22 binding CD22 B-cell cancers or B-cell related immune disorders scFv(s) (see e.g. Kawas S et al., AlAbs 3: 479-86 (2011)) CD25 binding CD25 various cancers of the B-cell lineage and immune scFv(s) disorders related to T-cells (see e.g. Muramatsu H et al., Cancer Lett 225: 225-36 (2005)) CD30 binding CD30 B-cell cancers orB-cell/T-cell related immune monoclonal disorders (see e.g. Klimka A et al., Br J Cancer antibody (ies) 83: 252-60 (2000)) CD33 binding CD33 myeloid cancer or immune disorder (see e.g. monoclonal Benedict C et al., J Immunol Methods 201: 223- 31 (1997)) antibody(ies)WO 2016/196344 PCT/US2016/034778 357 CD38 binding CD38 hematological cancers, B-cell related immune immunoglobul disorders, and T-cell related immune disorders in domains (see e.g. U.S. patent 8,153,765) CD40 binding CD40 various cancers and immune disorders (see e.g. scFv(s) EllmarkP et A., Immunology 106: 456-63 (2002)) CD45 binding CD45 Hematological cancers and myelodysplastic monoclonal syndromes (see e.g. Matthews D et al., Blood 94: 1237-47 (1999); Lin Y et al, Cancer Res 66: antibody (ies) and scFv(s) 3884-92 (2006); Pagel J et al, Blood 107: 2184- 91 (2006)) CD52 binding CD52 B-cell cancers, such as lymphoma and leukemia, monoclonal and B-cell related immune disorders, such as antibody (ies) autoimmune disorders (see e.g. U.S. Patent 7,910,104) CD56 binding CD56 immune disorders and various cancers, such as monoclonal lung cancer, Merkel cell carcinoma, myeloma (see e.g. Shin J et al., Hybridoma 18: 521-7 antibody (ies) (1999)) CD79 binding CD79 B-cell cancers or B-cell related immune disorders monoclonal (see e.g. Zhang L et al., Ther Immunol 2: 191- antibody(ies) 202 (1995)) CD 13 3 CD 133 various cancers, hematologic malignancies, and binding immune disorders (see e.g. Bidlingmaier S et al., monoclonal J Mol Med 86: 1025-32 (2008); Pavlon L et al., J antibodies and Microsc 231: 374-83 (2008); Rappa G et al., Stem Cells 26: 3008-17 (2008); Swaminathan S scFv(s) et al., J Immunol Methods 361: 110-5 (2010); Wang J et al., Hybridoma 29: 241-9 (2010); Zhu X et al., Mol Cancer Ther 9: 2131-41 (2010); Xia J et al., Sa Rep 3: 3320 (2013)) CD248 CD248 various cancers, such as inhibiting angiogenesis binding (see e.g. Zhao A et al., J Immunol Methods 363: scFv(s) 221-32(2011)) EpCAM EpCAM various cancers, such as ovarian cancer, binding malignant ascites, gastric cancer (see e.g. monoclonal Schanzer J et al., JImmunother 29: 477-88 antibody(ies) (2006)) PSMA binding PSMA prostate cancer (see e.g. Frigerio B et al., EurJ monoclonal Cancer 49: 2223-32 (2013)) anlibody(ies) Eph-B2 Eph-B2 various cancers such as colorectal cancer and binding prostate cancer (see e.g. AbengozarM et al., monoclonal Blood 119: 4565-76 (2012)) antibody(ies) Endoglin Endoglin various cancers, such as breast cancer and binding colorectal cancers (see e.g. Volkel T et al., monoclonal Biochim Biophys Res Acta 1663: 158-66 (2004)) antibody(ies) FAP binding FAP various cancers, such as sarcomas and bone monoclonal cancers (see e.g. Zhang J et al., FASEB J 27: 581-9 (2013)) antibody(ies) CEA binding CEA various cancers, such as gastrointestinal cancer, antibody (ies) pancreatic cancer, lung cancer, and breast cancer and scFv(s) (see e.g. NeumaierM et al., Cancer Res 50:2128-34 (1990);Pavoni E et al., BMC Cancer 6: 4 (2006); Yazaki P et al., Nucl Med Biol 35: 151­ 8 (2008); Zhao J et al., Oncol Res 17: 217-22 (2008)) various cancers such, as bladder cance (seer e.g.
CD24 binding CD24 monoclonal Kristians Gen et al., Lab Invest 90: 1102-16 antibody(ies) (2010)) LewisY LewisY various cancers, such as cervica cancerl and uterine cancer (see e.g. Power B et al., Protein antigen antigens binding Set 12: 734-47 (2003); Feridani A et al., scFv(s) Cytometry 71: 361-70 (2007)) adalimumab TNF-a various cancers and immune disorde suchrs, as rheumat oidarthri Crohntis, ’s Disease, plaque psoriasis, psoriati arthrc itankylosis, ing spondylit juveniis, leidiopathic arthri tis, hemolyti disec ase of the newborn afelimomab TNF-a various cancers and immune disorders ald518 IL-6 various cancers and immune disorders, such as rheumatoi arthrd itis anrukinzum ab IL-13 various cancers and immune disorders or ima-63 8 IL-12, IL-23 various cancers and immune disorders, such as briakinumab psoriasi rheumatoids, arthr itiinflas, mma tory bowel diseases, multiple sclerosis brodalumab IL-17 various cancers and immune disorders, such as inflamma torydiseases canakinumab IL-1 various cancers and immune disorders, such as rheumatoi arthrd itis various cancers and immune disorders, such as certolizumab TNF-a Crohn’s disease fezakinumab IL-22 various cancers and immune disorders, such as rheumat oidarthri psoriasistis, ganitumab IGF-I various cancers golimumab TNF-a various cancers and immune disorders, such as rheumat oidarthri psoriattis, arthriic tis, ankylosin spondylig tis infliximab TNF-a various cancers and immune disorde suchrs, as rheumatoi arthrid ankylositis, spondylitis,ng psoriati arthrc itpsoriis, asi Crohn's, diseass e, ulcerat coliive tis ixekizumab IL-17A various cancers and immune disorders, such as autoimmune diseases mepolizumab IL-5 . various immune disorders and cancers, such as B-cell cancers various cancers and immune disorders nerelimomab TNF-a olokizumab IL6 various cancers and immune disorders ozoralizumab TNF-a inflammation perakizumab IL17A various cancers and immune disorders, such as arthritis placulumab human TNF vario usimmune disorders and cancers sarilumab IL6 various cancers and immune disorders, such as rheumat oidarthr itankylosiis, spondylitng is siltuximab IL-6 various cancers and immune disorders -358-WO 2016/196344 PCT/US2016/034778 359 sirukumab IL-6 various cancers and immune disorders, such as rheumatoid arthritis BAFF tabalumab B-cell cancers ticilimumab or CTLA-4 various cancers tremelimumab IL23 immunologically mediated inflammatory tildrakizumab disorders tnx-650 IL-13 various cancers and immune disorders, such as B-cell cancers IL-6 receptor various cancers and immune disorders, such as tocilizumab or atlizumab rheumatoid arthritis ustekinumab IL-12, IL-23 various cancers and immune disorders, such as multiple sclerosis, psoriasis, psoriatic arthritis VEGFR, various cancer, such as breast cancer and colon Various growth EGFR, FGFR cancer, and to inhibit vascularization factors: VEGF, EGF1, EGF2, FGF Various IL-2R, IL-6R, various immune disorders and cancers cytokines: IL- IL-23R, 2, IL-6, IL-23, CD80/CD86, TNFRSF13/T CCL2, BAFFs, TNFs, NFRSFT7, RANKL TNFR Broadly־ Influenza viral infections (see e.g. Prabakaran P et al., neutralizing surface Front Microbiol 3: 277 (2012)) antibodies antigens (e.g. identified from bemaglutinins patient and matrix protein 2) samples Broadly Coronavirus viral infections (see e.g. Prabakaran P et al., neutralizing surface Front Microbiol 3; m (2012)) antibodies antigens identified from patient samples Various Filovirus viral infections (see. e.g. Olinger G et al., Proc antibodies surface Natl Acad Sci USA 109: 18030-5 (2012); Pettitt J antigens (e.g. et al., &7 TranslMed 5: 199rall3 (2013); VP35, VP40, Stahelin R, Expert Opm Ther Targets 18: 115-20 and (2014); Becquart P et al., PLoS One 9: 696360 glycoprotein) (2014); Stahelin R, Fron Microbiol 5: 300 (2014); TranE et al., J Virol 88: 10958-62 (2014); Murin C et al., Proc Natl Acad Sci USA 111: 17182-7 (2014)) Broadly Henipavirus viral infections (see e.g. Prabakaran P et al., neutralizing Front Microbiol 3: 277 (2012)) surface antibodies antigens identified from patient samples Various HIV surface viral infections (see e.g. Kitidee K et al., BMC Biotechnol 10: 80 (2010); YuL, Guan Y, Front antibodies antigens (e.g. including matrix protein Immunol 5: 250 (2014)) broadly Mapl7)neutralizing antibodies and scFvs 917. 917. 917. id="p-917" id="p-917" id="p-917" id="p-917" id="p-917" id="p-917" id="p-917" id="p-917" id="p-917" id="p-917"
[917] Whil esome embodiments of the inventio haven been described by way of illustrati it on,will be apparent that the invention may be put int opracti witce hmany modifications, variations and adaptati ons,and wit hthe use of numerous equivalents or alternati solutive ons that are withi then scope of persons skill edin the art, without departing from the spiri oft the inventio orn exceeding the scope of the claims. 918. 918. 918. id="p-918" id="p-918" id="p-918" id="p-918" id="p-918" id="p-918" id="p-918" id="p-918" id="p-918" id="p-918"
[918] All publications, patent ands, pate ntapplicati onsare herei incorpn orate by d reference in their entiret to ythe same extent as if each individu publal icatio patentn, or patent applicati wason specifical andly individua indilly cated to be incorpora ted by reference in its entirety. The internati pateonal ntapplicati publion cations WO 2014/164680, WO 2014/164693, WO 2015/138435, WO 2015/138452, WO 2015/113005, WO 2015/113007, and WO 2015/191764, are eac hincorporat ed herei byn reference in its entire ty.The disclosure of U.S.s patent applicatio ns US20150259428; 62/168,758; 62/168,759; 62/168,760; 62/168,761; 62/168,762; and 62/168,763 are each incorpora hereited byn reference in its entire ty.The disclosure of WO 2016/126950 is incorpora hereited byn reference in its entire ty.
The complet discle osures of all electronicall avaiylabl biologicale sequence informati fromon GenBank (National Cente forr Biotechnol Informatiogy U.S.)on, for amino aci dand nucleoti sequencesde cite hereid aren each incorpora hereited n by reference in their entirety. -360-WO 2016/196344 PCT/US2016/034778 361 Sequence Listing ID Number Text Description Biological Sequence SEQ ID NO: 1 Shiga-like toxin 1 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT Subunit A (SET- SLLMIDSG SGDNLF AVD VRGIDPEEGRFNNLRLIV 1A) ERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTA VTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQ1 QRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLS S VLPDYHGQDSVRVGRISFGS1NAILGS VALILNCH HHASRVARMASDEFPSMCPADGRVRGITHNK1LW DSSTLGAILMRRTISS Shiga toxin SEQ ID NO:2 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT Subunit A SLLMIDSGTGDNLFAVDVRGIDPEEGRFNNLRLIV ERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTA VTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQI QRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLS SVLPDYHGQDSVRVGRISFGSINAILGSVAL1LNCH HHASRVARMASDEFPSMCPADGRVRGITHNKILW DSSTLGAILMRRTISS SEQ ID NO:3 Shiga-like toxin 2 deftvdfssqksyvdsensirsaistplgnisqggv Subunit A (SET- SVSVINHVLGGNYISLNVRGLDPYSERFNHLRLIM 2A) ERNNLYVAGFLNTETNIFYRFSDFSHISVPDVITVS MTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLDL MEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQR GFRPALSEASPLYTMTAQDVDLTLNWGRISNVLP EYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGS YSVRSVSQKQKTECQIVGDRAAIKVNNVLWEAN TIAALLNRKPQDLTEPNQ SEQ ID NON Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT effector SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIV polypeptide SET- ERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTA 1A-WT VTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQI QRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLS SVLPDYHGQDSVRVGRISFGSINAILGSVALILNCH HHASRVAR SEQ ID NO:5 Shiga toxin KEFTLDFSTAK'n'VDSLNMRSAIGTPLQTISSGGT effector SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIV polypeptide SET- ERNNI_.Y\?TGF\7NR_TNNVFYRFADFSHVTFPGTTA VTL SGDS SYTTLQR VAGISRTGMQINRHSLTTS YL 1A-FR DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQI QRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLS SVLPDYT-IGQDSVRVGRISFGSINAILGS VALILNCH HHASAVAA SEQ ID NO:6 Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTTSSGGT effector SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLTV polypeptide SET- ERNNI.YVTGFY'NRTNNWWFADFSHVTFPGTNI, lA-comboO VPMVATVSYTTLQRVAGISRTGMQINRHSLTTSY LDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQ IQRGFRTTI..DDLSGRSY'WTAEDVDLTLNWGRLS SVLPDYHGQDSVRVGRISFGSINAILGSVALILNCH HHASAVAA SEQ ID NO:7 Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT effector SLLMIDSGSGDNLFAVDILGFDFTLGRFNNLRLIVEWO 2016/196344 PCT/US2016/034778 362 polypeptide SET- RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV lA-combol TLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVrvrAEALRF’RQIQ RGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVAL1LNCH HHASAVAA SEQ ID NO:8 Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT effector SIJ.AnDSGIGDNLFAVDILGFDFn.GRFNNLRLIVE polypeptide SET- RNNLYATGFVNRTNNWA’RFADFSHWFPGTTAV lA-combo2 TLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQ RGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH HASAVAA SEQ ID NO:9 Shiga toxin KEF 1LDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT SLLMIDSGSGDNI,FAV’NLWMVATVGRFNNI..RLI effector polypeptide SET- VERNNLYVTGFVNRTNNVFYRFADFSHVTF^^ AVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSY lA-combo3 LDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQ IQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLS SVLPDYHGQDSVRVGRISFGSINAILGSVALILNCH HHASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSATGTPLQTISSGGT NO: 10 effector SLLMTDNLVPMVATWDVRGTDPEEGRFNNLRLIV polypeptide SET- FRNNLYVTGFVNRTNTWFYRFADFSHVTFPGTTA lA-combo4 VTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQI QRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLS SVLPDYHGQDSVRVGRISFGSINAILGSVALILNCH HHASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT effector NO: 11 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SET- RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1 A־combo5 TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQ RGFR1 ‘ 1LDDLSGAS YVMTAEDVDLTLNWGRLS S VLPDYHGQDSVRVGRISFGSINAILGSVALILNCH HHASRVAR SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 12 effector SLLNflDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SET- RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV lA-combo6 TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQ RGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRTSFGSTNAILGSVALILNCH HHASRVAR SEQ ID Shiga toxin KEFTLDFSTAKIYVDSLNVIRSAIGTPLQTISSGGT NO: 13 effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SET- RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV lA-combo7 TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQ RGFR1" 1LDDLSGAS YVMT AED VDLTLN WGRLS S VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH HASAVAAWO 2016/196344 PCT/US2016/034778 363 SEQID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 14 effector SLLMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIVE polypeptide SET- RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV TLS/XDSSYTTLQRVAGISRTGMQINRHSLTTSYLD lA־combo8 LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQ RGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVAL1LNSHH HASAVAA SEQID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 15 effector SLLMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVE polypeptide SET- RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV lA-combo9 TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQ RGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH HASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 16 SLEMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE effector polypeptide SET- RNNIAAGXiFVNRTNNVFYRFADFSHVTFPGTTAV lA-combolO TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSATSLTQSVARAMLRFVrvrAEALRF’RQIQ RGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH HASAVAA SEQID Sbiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 17 effector SIJ.AnDSGIGDNLFAVGILGFVFTLGRFNNLRLIVE polypeptide SET- RNNLYATGFVNRTNNWA’RFADFSHWFPGTTAV lA-combol 1 TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLnQSVARAAn,RFVTVTAEALRFRQIQ RGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH HASAVAA SEQ ID Shiga toxin AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 18 effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SET- RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV lA־combol2 TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQ RGFRriLDDLSGRSYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH HASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSATGTPLQTISSGGT NO: 19 effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLTVE polypeptide SET- RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV lA-combol3 TLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQTQ RGFRGILGDVFTRSYVMTAEDVDLTLNWGRLSSV LPDYHGQDSVR.VGRISFGSINAILGSVALILNSHHH ASAVAA SEQID Shiga toxin KEFTLDFSTAKIYVDSLNVIRSAIGTPLQTISSGGT NO:20 effector SLLM1DSG1GDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SET- RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1 A-combol4 TLSADSSYTTLQRVAGTSRTGMQINRHSLTTSYLD LMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQ RGFR1" 1LDDLSGASYVMTAEDVDLTLNWGRLSSWO 2016/196344 PCT/US2016/034778 364 VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH HASAVAA SEQ ID Shiga toxin AEFTLDFSTAKTYVDSLNVIRSATGTPLQTISSGGT NO:21 effector SLLMTDSGIGDNLFAVDILGFDFTLGRFNNLRLTVE polypeptide SET- RNNI^AYGFVNRTNNVTO lA״combol5 TESADSSY^TLQRVAGISRTGMQINRHSETTSYED EMSHSATSI.YQSVARAW,RFVTVTAEALRFRQIQ RGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH HASAVAA SEQ ID Shiga toxin KEFILDFSTAKTYVDSLNVTRSATGTPLQTISSGGTS NO:22 effector LLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVER polypeptide SET- NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVT 1 A־combol6 LSADSSYTTLQRVAGTSRTGMQINRHSLTTSYLDL MSHSATSLTQSVARAMLRFVTVTAEALRFRQIQR GFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSV LPDYHGQDSVRVGRISFGSINAILGSVALILNSHHH ASAVAA SEQ ID Shiga toxin AEFILDFSTAKTYVDSLNV1RSAIGTPLQTISSGGTS NO:23 effector LLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVER polypeptide SET- NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVT lA-combol7 LSADSSYTTLQRVAGISRTGMQTNRHSLTTSYLDL MSHSATSLTQSVARAMLRFVTVTAEALRFRQTQR GFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSV LPDYHGQDSVRVGRISFGSINAILGSVALILNSHHH ASAVAA SEQ ID Shiga toxin AEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO:24 effector LLM1DSGIGDNLFAVNLVPMVATVGRFNMLRLIV polypeptide SET- ERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTA IA-combol8 VTLSADSSYTTLQRVAGISRTGMQ1NRHSLTTSYL DLMSHSATSLTQSVARAMLRFVTVTAEALRFRQ1 QRGFRTrLDDLSGASYVMTAEDVDLTLNWGRLS SVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HH ASAVAA SEQ ID Shiga toxin KEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO:25 effector LLMIDSGIGDNLFAVDVRGIAPIEARFNNLRLIVER polypeptide SET- NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVT lA-combol9 LSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDL MSHSATSLTQSVARAMLRFVTVTAEALRFRQIQR GFRTTLAALSGASYVMTAEDVDLTLNiWGRLSSV LPDYHGQDSVRVGRISFGSINAILGSVALILNSHHH ASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO:26 effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SET- RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV lA-combo20 TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSATSLTQSVARAMLRFVTVTAEALRFRQ1Q RGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSS A'LPDYHGQDSVRVGRISFGSINAILGSVALILNSHH HARNLVPMVATVASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO:27 effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SET- RNNLYVTGFVNATNNVFYRF/ADFSHVTFPGTTAV lA-combo21 TLS/XDSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVrvrAEALRF’RQIQWO 2016/196344 PCT/US2016/034778 365 RGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH HASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSATGTPLQTISSGGT NO:28 effector SLLMIDSGSGDNLFAVDVRGIAPEEGRFNNLRLIV polypeptide SET- ERNTO;YVTGFVNTITNNVFYRFADFSHVTTPGT^^ lA-combo22 VTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYL DLMSHSATSLTQSVARAMLRFVTVTAEALRFRQI QRGFRTTLDDLSGASYVMTAEDVDETLNWGRLS S VLPDYHGQDSVRVGRISFGSINAILGS VALILNSH HHASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO:29 effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SET- RNNLYVTGFVNRTNNAFYRFADFSHVTFPGTTAV 1 A־combo23 TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQ RGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH HASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO:30 effector SLLMTDSGIGDNLFAVDILGFDFTLGRFNNLRLTVE polypeptide SET- RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV lA-combo24 TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQ RGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH HASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT N0:31 effector SLLM1DSGIGDNLFAVDILGFDFTLGRFNNLRL1VE polypeptide SET- RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV lA-combo25 TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLA LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQ RGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH HASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO:32 effector SLLNflDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SET- RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV lA-combo26 TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQ RGFRTTLDDLSGASYVMTAEDVALTLNWGRLSS VLPDYHGQDSVRVGRTSFGSINAILGSVALILNSHH HASAVAA SEQ ID NO:33 scFv-3::SLT-lA- MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYN WT VHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGK ATLTADKSSSl'VYMQLSSLTSEDSAVYYCARSNYY GSSYVWFFDVWGAGTrVTVSSGSTSGSGKPGSGEG SQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWY QQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYS LTISRVEAEDAATYYCQQWISNPP^GAGl ’KLELKE FPKPSTPPGSSGGAPKEFTLDFSTAKTYVDSLNVIRS AIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDP EEGRFNNLRLIVERNNLYVTGFV^TNNVFYRFADF SHVTFPGTTAV'TLSGDSSYTn.QRVAGISRTGMQIN RHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAWO 2016/196344 PCT/US2016/034778 366 EALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLN WGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALI LNCHHHASRVAR SEQ ID NO :34 SLT-IA-FR::scFv- MKEFTLDFSTAKTYVDSLNVIRSATGTPLQTTSSGGT 1 SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVER NNT.Y\YGFVTNR.TNNWYRFADFSHV’TFPGTTAY7TL, SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAAH.RFYTYTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNP YTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWR GGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID NO:35 SLT-IA-FR::scFv- AflSEFHJJFSTAKTYWSIJ^RSAIGTPLQTISSGGT 2 SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVER NNLYVTGFWTITNNWYRFADFSIIVTFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVAR.AMLRFVTVT.'XE.'XLRF'RQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSV2ALILNCHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNP YTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQE SGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPG RGLEWTGVMWRGGSTDYNAAFMSRLNTTKDNSKN QVSLRLSSVTAADTAVYYCAKSMTTTGFVMDSWG QGSLVTVSSA SEQ ID NO :36 MKEMLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT SLT-lA-FR::scFv- 4 SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVER NNLYVrGFVNRl'NNVFYRF/ADFSHVIFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINA1LGSVALILNCHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CRASQDVNTAVAWYQQKPGKAPKLLTYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTT PPTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGG GGSEVQLVESGGGLVQPGGSLRLSCAASGFNTKDTY IHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRF TISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGD GFAAAMDAAVGQGTI..VTVSSA SEQ ID NO:37 MKEFTLDFSTAKTYVDSLN^RSAIGTPLQTISSGGT SLT-lA-FR::scFv- SLLMIDSGSGDNLFAVDWGIDPEEGRFNNLRLIVER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGTSRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQTQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITWO 2016/196344 PCT/US2016/034778 367 CRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYP Y TFGQGTKLEIKGGGGSQ VQL VQSG AE VKKPG AS V KVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWL HADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRS EDTAVYYCARER1QLWFDYWGQGTLVTVSS SEQ ID NO:38 SLT-lA-FR::scFv- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT 6 SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVER NTOYAYGFWRTNNWYRFADFSHVTTPGTTAY^ SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASAVAAE FPKPSTPPGS SGG APE VQL V ESGGG LVQ PGG SLRL S CAASGFTFSDS WIHWVRQAPGKGLE WVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDT AVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQ QKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK SEQ ID NO:39 MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT SLT-lA-FR::scFv- 7 SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVFL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRS Y VM FAED VDLTLN W GRL S S V LPD YHG QDSVRVGRISFGSINAILGSVALILNCHHHASAVAAE FPKPSTPPGSSGGAPDIV'LY’QSPASLAVSLGQRATISC RATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSV DSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQS RRVPYTFGGGTKLETKGGGGSEVQLQQSGPELVKPG ASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWTG YVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELS SLTSEDSAVYYrCARQAWGYPWGQGTLVTVSA SEQ ID NO:40 SLT-lA-FR::scFv- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT 8 SLLMIDSGSGDNLFAV'DVRGIDPEEGRFNNLRLIVER NNLYVI’GFVNRl’NNVFYRFADFSHV'H^PGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRTSFGSINAILGSVALILNCHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQTTSSLSASLGDRVTISC RASQDISNYLAWYQQKPDGTVKLLIYYTSILHSGVP SRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLP WTFGCGTKLEIKGSTSGSGKPGSGEGSEVQLVESGG GLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKC LEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLY LQMSSLKSEDTAMYYCARHSGYGTHWGVLFAYW GQGTLVTVSA SEQ ID N0:41 SLT-lA-FR::scFv- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT 9 SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLTVER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGTSRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQTQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGWO 2016/196344 PCT/US2016/034778 368 QDSVRVGRISFGSINAILGSVALILNCHHHASAVAAE FPKPSTPPGSSGGAPDIELTQSPSSFSVSLGDRVTITC KASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVP SRFSG SGSGKD YTL SITSLQTED VATY YCQQYWSTP TFGGGTKLE1KGSTSGSGKPGSGEGSKVQLQESGPS LVQPSQRLS1TCTVSGFSLISYGVHWVRQSPGKGLE WLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFK MN SLQADDIAIYF C• AKTLIT TGY AMD Y־W GQGTT V TVSS SEQ ID NO:42 SLT-lA-WT::scFv- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT 9 SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVER NNLYVrGFVNRl’NNVFYRFADFSHV’IFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASRVARE FPKPSTPPGSSGGAPDIELTQSPSSFSVSLGDRVTITC KASED1YNRLAWYQQKPGNAPRLL1SGATSLETGVP SRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTP TFGGGTKLETKGSTSGSGKPGSGEGSKVQLQESGPS LVQPSQRLSTTCTVSGFSLISYGVHWVRQSPGKGLE WLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFK MNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTV TVSS SEQ ID NO:43 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo l::scFv-l SLLMIDSGSGDNLFAVDILGFDFTLGRFNNLRLIVER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFTISSLQPEDIATT'YCQQYWSNP YTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWR GGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID NO:44 SLT-1A- MKEFTLDFSTAKTYVDSLNV1RSAIGTPLQTISSGGT combo7::scFv-1 SLLMTDSGIGDNLFAVDILGFDFTLGRFNNLRLIVER ^fNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRISFGSTNAILGSVALILNSHHHASAVAA EFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNP YIF GQGTKVEIKGGGG SQ VQLQESGP GLVRP SQTL S LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWR GGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVA'YC ׳\KSMITTGF VMD S W GQG SL VT VS S SEQ ID NO:45 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSATGTPLQTTSSGGT comboS: :scFv-1 SLLMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIVER NNT..Y\,TGFVTNR.TNNVFYRFADFSHV’TFPGTTA\7TL,WO 2016/196344 PCT/US2016/034778 369 SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLl^SVARAMLRFVTVTAEALRFRQIQRG TLDDL SG ASYVMTAED VDLTLNWGRL SS VLPDYH GQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA EFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVT1T CKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNP YTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWR GGSTDYNAAFMSRLNTTKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID NO:46 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT SLLMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVER combo9::scFv-l NNLYVrGFVNRrNNVFYRFADFSHV’IFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA efpkpstppgssggapdiqmtqspsslsasvgdrvtit CKASEDTYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTUFTFTISSLQPEDIATYYCQQYWSNP YTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWR GGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID NO:47 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combolO::scFv-l SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSATSLTQSVARAMLRFVTVTAEALRFRQTQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNSHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTTT CKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNP YTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWR GGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID NO:48 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT SLLMTDSGIGDNLFAVGILGFVFTLGRFNNLRLIVER combo 1 l::scFv-l ^fNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA EFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFnSSLQPEDIATYYCQQYWSNP Y TFGQGTKVEIKGGGG SQVQLQESGPGL VRPSQTL S LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWR GGSTDYTsfAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYC/XKSMITTGFVMDSWGQGSLVrVSS SEQ ID NO:49 SLT-1A- MAEFTLDFSTAKTYVDSLNVIRSATGTPLQTTSSGGT combo 12 ::scFv-1 sllmidsgigdni,favdilgfdfti,grfnni,rliyt;rWO 2016/196344 PCT/US2016/034778 370 NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAXTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSATSLl^SVARAMLRFVTVTAEALRFR TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGS1NAILGSVALILNSHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNP YTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWR GGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID NO :50 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo 14 ::scFv-1 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVER NNLYVrGFVNRlNNVFYRF/ADFSHVIFPGTTAVTL SADSSYTTLQRVAG1SRTGMQINRHSLTTSYLDLMS HSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA EFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTTT CKASEDTYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNP YTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWR GGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ1DNO:51 SLT-1A- MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRL1VER combo 15 ::scFv-1 NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGTSRTGMQINRHSLTTSYLDLMS HSATSLTQSVARAMLRFVTVTAEALRFRQTQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRTSFGSINAILGSVALTLNSHHHASAVAA EFPKPSTPPGSSGGAPDTQMTQSPSSLSASVGDRVTIT CKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNP YTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWR GGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID NO :52 SLT-1A- MKEFILDFSTAKTYVDSLNVIRSATGTPLQTTSSGGTS combo 16: :scFv-1 LLMTDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERN NLY\'TGFV7NRTNNWYRFADFSHVTFPGTTAX,TI...S ADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSH SATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNSHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNP Y TFGQGTKVEIKGGGG SQVQLQESGPGL VRPSQTL S LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWR GGSTDYTsfAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSSWO 2016/196344 PCT/US2016/034778 371 SEQ ID NO:53 SLT-1A- MAEFILDFSTAKTYVDSLNV1RSAIGTPLQTISSGGTS LLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERN combo 17 ::scFv-1 NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS ADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSH SATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNSHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFT1SSLQPEDIATYYCQQYWSNP YTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWR GGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID NO :54 SLT-1A- MAEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS combo 18: :scFv-1 LLMIDSGIGDNLFAVNLVPMVATVGRFNNLRLIVER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAG1SRTGMQINRHSLTTSYLDLMS HSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA EFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVT1T CKASED1YNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDF'iFlISSLQPEDIATYYCQQYWSNP YTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWR GGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID NO :55 SLT-1A- MKEFILDFSTAKTYVDSLNVIRSA1GTPLQTISSGGTS combo 19: :scFv-1 LLMIDSG1GDNLFAVDVRGIAP1EARFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS ADSSYTTLQRVAG1SRTGMQINRHSLTTSYLDLMSH SATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LAALSGASYVMTAEDVDLTLNWGRLSSWPDYHG QDSWVGRISFGSINAILGSVALILNSHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNP YTFGQGTKVTJKGGGGSQVQLQESGPGLWPSQTLS ltctvsgfsltsygvi-iwvrqppgrglewigvmwr GGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID NO :56 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo20:: scFv-1 SLLM1DSGIGDNLFAVDILGFDFTLGRFNNLRLIVER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNSHHHARNLVPM VATVASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSL SASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKL LIS GAT SLETG VP SRI׳' S G S GS G TDFTFTI SSL QPEDI AT YYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQES GPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGR GLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQWO 2016/196344 PCT/US2016/034778 372 VSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVI vss SEQ ID NO :57 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSATGTPLQTTSSGGT comboO::scFv-2 SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLTVER NNT..Y\,TGFVTNR.TNNWYRFADFSHV’TFPGTNIA'TM VATVSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARANRRFWWAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSWVGRISFGSINAILGSVALILNCHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNP YTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQE SGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPG RGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKN QVSLRLSSVTAADTAVYYCAKSM1TTGFVMDSWG QGSLVTVSSA SEQ ID NO :58 SLT-1A- MKEFTLDFSTAKTY\T)SLN\7IRSAIGTPLQTISSGGT combo2::scFv-2 SL.LMIDSGIGDNI.FAVDILGFDFTL.GRFNNI.RLIW.R NNLYVTGFWTITNNWYRFADFSI1VTFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVT.'XE.'XLRF'RQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNSHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNP YTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQE SGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPG RGLEWTGVMWRGGSTDYNAAFMSRLNTTKDNSKN QVSLRLSSVTAADTAVYYCAKSMTTTGFVMDSWG QGSLVTVSS SEQ ID NO :59 MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT SLT-1A- combo3::scFv-2 SLLMIDSGSGDNLFAVNLVPMVATVGRFNNLRLIVE RNNLYVTGFVNRTNNVFYRF'ADFSFIVrFPGTl'AVT LSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLM SHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFR TTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRISFGSINAILGSVAL1LNCHHHASAVA AEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVT TTCKASEDTYNRLTWYQQKPGKAPKLLISGATSLET GVPSRFSGSGSGTDFTFTTSSLQPEDIATYYCQQYWS NPYTFGQGTKVETKGSTSGSGKPGSGEGSTK.GQVQL QESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQP PGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNS KNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDS WGQGSLVTVSSA SEQ ID NO:60 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo4::scFv-2 SLLMIDNLVPMVATVVDVRGIDPEEGRFNNLRLIVE RNNLYVTGFVNRTNMVFYRFADFSHVTFPGTTAVT LSGDSSYTTLQRVAGTSRTGMQINRHSLTTSYLDLM SHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFR TTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRTSFGSINAILGSVALTLNCHHHASAVA AEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTWO 2016/196344 PCT/US2016/034778 373 ITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLET GVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWS NPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQL QESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQP PGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNS KNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDS WGQGSLVTVSSA SEQ ID NO:61 SLT-1A- MKEF؛LDFSTAKTYAT)SLNV؟RSAIGTPLQTISSGGT combo 10 ::scFv-2 sllmidsgigdni,favdilgfdfti,grfnni,rlivt;r NNLYVrGFVNRl’NNVFYRFADFSHV’IFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNSHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNP YTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQE SGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPG RGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKN QVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSSA SEQ ID NO:62 SLT-1A- MKEFTLDFSTAKTYVDSLNV1RSAIGTPLQTISSGGT combo 13 ::scFv-2 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRG 1LGD VFTRSY VMTAED VDLTLN WGRLS S VLPD YHG QDSVRVGRISFGSINAILGSVALILNSHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTTT CKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFIFTISSLQPEDIATYYCQQYWSNP YTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQE SGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPG RGLEWIGV’MWGGSTDY'NAAFMSRLNITKDNSKN QVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSS SEQ ID NO:63 SLT-1A- MKEFTLDFSTAKTYVDSLNV1RSAIGTPLQTISSGGT combo22:: scFv-2 SLLMIDSGSGDNLFAVDVRGIAPEEGRFNNLRLIVER ^fNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA EFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTTT CKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFnSSLQPEDIATYYCQQYWSNP YTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQE SGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPG RGLEWIGVMWRGGSTDYNA2YFMSRLNITKDNSKN QVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSSA SEQ ID NO:64 scFv-3::SLT-lA- MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYN combo5 VHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGK ATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYYWO 2016/196344 PCT/US2016/034778 374 GSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEG SQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWY QQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYS LTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKE FPKPSTPPGSSGGAPKEFTLDFSTAKTYVDSLNVIRS A1GTPLQTISSGGTSLLM1DSGIGDNLFAVDILGFDFT LGRFNnLrEIVERNNLYVTGFVNRTNNVFYRFADFS HVTFPGTTAVTLSADSSY1TLQRVAGISRTGMQINR HSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAE ALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLN WGRLSSVLPDYHGQDSVRVGRISFGSTNAILGSVALI LNCHHHASRVAR SEQ ID NO:65 scFv-3::SLT-lA- MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYN combo6 VHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGK ATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYY GSSYVWFFDVWGAGTrVTVSSGSTSGSGKPGSGEG SQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWY QQKPGSSPKPW1YATSNLASGVPARFSGSGSGTSYS LT1SRVEAEDAATYYCQQWISNPPTFGAGTKLELKE FPKPSTPPGSSGGAPKEFTLDFSTAKTYVDSLNVIRS ATGTPLQTISSGGTSLLMTDSGIGDNLFAVDILGFDFT LGRFNNLRLTVERNNLYVTGFVNRTNNVFYRFADFS HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINR HSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAE ALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLN WGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALI LNCHHHASRVAR SEQ ID NO:66 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo7::scFv-4 SLLM1DSGIGDNLFAVDILGFDFTLGRFNNLRL1VER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGTSRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFWWAEALRFRQTQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRTSFGSINAILGSVALTLNSHHHASAVAA EFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTT PPTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGG GGSEVQLW,SGGGLVQPGGSLRLSCAASGFNIKDlAr IHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRF TISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGD GFYAMDYWGQGTLVTVSS SEQ ID NO:67 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo!4::scFv-4 SLLMTDSGIGDNLFAVDILGFDFTLGRFNNLRLIVER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTH.QRVAGISRTGMQINRHSLTTSYLDLMS HSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA EFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTT PPTFGQGTKVEIKGGG G SGGG G SGGGG SGGGG S GG GGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTY IHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFWO 2016/196344 PCT/US2016/034778 375 TISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGD GFYAMDYWGQGTLVTVSS SEQ ID NO:68 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSATGTPLQTTSSGGT combo7::scFv-5 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVER NTOYYTGFVTTITNNWYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA efpkpstppgssggapdiqmtqspsslsasvgdrvtit CRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYP YTFGQGTKLEIKGGGGSQVQLVQSGAEVKkPGASV K VS CK A S GYTFT SYD VH W VRQ AP GQRLE WMG WL HADTGITKFSQKF'QGRVnTRDTSASlAYVlELSSLRS EDTAVYYCARERIQLWFDYWGQGTLVTVSS SEQ ID NO:69 SLT-1A- MKEFTLDFSTAKTYATISLNVTRSAIGTPLQTISSGGT comboS: :scFv-5 SLLMIDSGIGDNLFzWDILGFVFTLGRFNNLRLIVER NTVLYVTGFVNTITNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVAR.AMLRFVTVT.'XE.'XLRF'RQIQRGFRT TLDDL SG ASYVMTAED VDLTLNWGRL SS VLPDYH GQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA EFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGV PSRFSGSGSGTDFTLT1SSLQPEDFATYYCQQYNSYP YTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASV KVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWL HADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRS EDTAVYYCARERIQLWFDYWGQGTLVTVSS SEQ ID NO :70 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo9: :scFv-5 SLLMIDSGIGDNLFAVGILGFDFTLGRF'NNLRLIVER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSV'RVGRISFGSINAILGSVALILNSHHHASAVAA efpkpstppgssggapdiqmtqspsslsasvgdrvtit CRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGV PSRFSGSGSGTDFTLTTSSLQPEDFATYYCQQYNSYP YTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASV KVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWL HADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRS EDTAVYYCARERIQLWFDYWGQGTLVTVSS SEQ ID NO:71 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT comboil ::scFv-5 SLLMIDSGIGDNLFAVGILGFVFTLGRFNNLRLIVER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRTSFGSINAILGSVALTLNSHHHASAVAA EFPKPSTPPGSSGGAPDTQMTQSPSSLSASVGDRVTIT CRASQGISSWLAWYQQKPEKAPKSLTYAASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYP YTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVWO 2016/196344 PCT/US2016/034778 376 KVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWL HADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRS EDTAVYYCARERIQLWFDYWGQGTLVTVSS SEQ ID NO:72 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSATGTPLQTTSSGGT combo7::scFv-6 sllmidsgigdni,favdilgfdfti,grfnni,rli\t;r NNI.Y\YGFVTNR.TNNWYRFADFSHV’TFPGTTA\7TL, SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAAn.RFWWAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA EFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLS CAASGFTFSDS WIHWVRQAPGKGLE WVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDT AVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQ QKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYLYHPATFGQGIKVEIK SEQ ID NO:73 SLT-1A- MKEFTLDFSTAKTYAT)SLN\7IRSAIGTPLQTISSGGT combo8::scFv-6 SLLMIDSGIGDNI.FAVDILGFWnXiRFNNI.RLIW.R NNI.YVTGFVNRTNNWWFADFSI-IVTFPGTTAXGI, SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLl^SVARAMLRFVTVTAEALRFRQI^^ TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA EFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLS CAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDT AVYYCARRHWPGGFDYWGQGTLVT'VSSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQ QKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK SEQ ID NO :74 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT SLLMIDSGIGDNLFAVGILGFDFTLGRF'NNLRLIVER combo9::scFv-6 NNLYVrGFVNRrNNVFYRF/ADFSHVIFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT ILDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSWVGRISFGSINAILGSVALILNSHHHASAVAA EFPKPSTPPGSSGGAPEVQLYTSGGGLVQPGGSLRLS CAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYTQMNSLRAEDT AVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQ QKPGKAPKLLTYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK SEQ ID NO:75 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo21::scFv-6 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVER NNLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA EFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLS CAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTWO 2016/196344 PCT/US2016/034778 377 AVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDI QMTQSPSSLSASVGDRVIITCRASQDVSTAVAWYQ QKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK SEQ ID NO :76 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo23::scFv-6 sixmidsgigdnlfavimlgfdftlgrfnnlrliwji NNIA'AG'GFWRTNNAFYRFADFSHVTTPGTTA^ SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARANH.RFYTYTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA EFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLS CAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDT AVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQ QKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK SEQ ID NO:77 SLT-1A- MKEFTLDFSTAKTYAT)SLN\7IRSAIGTPLQTISSGGT SL.LMIDSGIGDNI.FAVDILGFDFn.GRFNNI.RLIW.R combo24::scFv-6 NNLYVTGFVNRTNNVFYRF'ADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLl^SAARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA EFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLS CAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDT AWYCARRHWPGGFDYWGQGTLVT'VSSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQ QKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK SEQ ID NO :78 MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT SLT-1A- combo25::scFv-6 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVER NNLYVrGFVNRl'NNVFYRF/XDFSHVIFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMS HSGTSLTQSVARAMLRFVTVtAEALRFRQlQRGFRT ILDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA EFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLS CAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTA^QMNSLRAEDT AVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQ QKPGKAPKLLTYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK SEQ ID NO:79 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo26::scFv-6 SLLM1DSGIGDNLFAVDILGFDFTLGRFNNLRLIVER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQ1NRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQTQRGFRT TLDDLSGASYVMTAEDVALTLNWGRLSSVLPDYH GQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA EFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLS CAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGWO 2016/196344 PCT/US2016/034778 378 STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDT AVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDI QMTQSPSSLSASVGDRVIITCRASQDVSTAVAWYQ QKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK SEQ ID NO :80 SLT-1A- MKEFl'LDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT comboO::scFv-8 SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVER NNT..Y\,TGFVTNR.TNNWYRFADFSHVTFPGTNIA'TM VATVSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQTTSSLSASLGDRVTISC RASQDISNYLAWYQQKPDGTVKLLIYYTSILHSGVP SRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLP WTFGCGTKLEIKGSTSGSGKPGSGEGSEVQLVESGG GLVKPGGSLKLSCAASGFAFS1YDMSWVRQTPEKC LEWVAYISSGGGTTYYPDTVKGRFT1SRDNAKNTLY LQMSSLKSEDTAMYYCARHSGYGTHWGVLFAYW GQGTLVTVSA SEQ ID NO:81 SLT-1A- MKEFl'LDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo7::scFv-7 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSY^ i 1 Lt^lR-A .A.(jISR-1GMt^HStRFISLI Ik^YLDL^lS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRISFGS1NAILGSVALILNSHHHASAVAA EFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATIS CRATES VEYYGTSLVQWYQQKPGQPPKLL1YAASS VDSGVPARFSGSGSGTDFSLTTHPVEEDDIAMYFCQ QSRRWYTFGGGTKLEIKGGGGSEVQLQQSGPELVK PGASVKMSCKASGYTFTSYVMHWVKQKPGQGLE WIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYM EL S SLTSED S AVYY CARQ A W GYPW GQGTL VT VS A SEQ ID NO :82 SLT-1A- MKEFT'LDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo7::aCD38- SLLMIDSGIGDNLFAVDILGFDFTLGRF'NNLRLIVER scFv-1 NNLYVI’GFVNRTNNVFYRFADFSHV'H^PGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYH GQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA EFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTTT CKASEDTYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNP YTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWR GGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVAn/CAKSMITTGFVMDSWGQGSLWVSS SEQ ID NO :83 heavy chain ABR1 YTFTSYV'MH SEQ ID NO :84 heavy chain ABR2 WIGYTNPYNDGTKY SEQ ID NO :85 heavy chain ABR3 kGTYYYGSRVFDY SEQ ID NO :86 light chain ABR1 KSLLNSNGNTYLY SEQ ID NO :87 light chain ABR2 LLTYRMSNLAS SEQ ID NO :88 light chain ABR3 MQHLEYPF SEQ ID NO:89 heavy chain ABR1 YAFSSYWMNWO 2016/196344 PCT/US2016/034778 379 SEQ ID NO :90 heavy chain ABR2 WIGQIWPGDGDTNY SEQ ID NO:91 heavy chain ABR3 RRETTTVGRYYYAMDY SEQ ID NO:92 light cliain ABR1 QSVDYDGDSYLN SEQ ID NO :93 light cliain ABR2 LLIYDASNLVS SEQ ID NO :94 light chain ABR3 QQSTEDPW SEQ ID NO :95 heavy chain ABR2 WIGQIWPGDGDTNYNG SEQ ID NO :96 heavy cliain ABR1 GSISTSGMGVG SEQ ID NO :97 heavy chain ABR2 WIGHIWWDDDKRY SEQ ID NO :98 heavy chain ABR3 RMELWSYYFDY SEQ ID NO:99 light chain ABR1 SSVSYMH SEQ ID NO :100 light chain ABR2 LL1YDTSKLAS SEQ ID NO :101 light chain ABR3 FQGSVYPF SEQ ID NO :102 heavy chain CDR1 GYTFTSYNMH SEQ ID NO: 103 heavy chain CDR2 AIYPGNGDTSYNQKFKG SEQ ID NO :104 heavy chain CDR3 AQLRPNYWYFDV SEQ ID NO: 105 light chain CDR1 RASSSVSYMH SEQ ID NO: 106 light chain CDR2 ATSNLAS SEQ ID NO :107 QQWISNPPT light cliain CDR3 SEQ ID NO :108 heavy cliain CDR1 GYTFTSYNVH SEQ ID NO: 109 heavy chain CDR3 SNYYGSSYVWFFDV SEQ ID NO: 110 RASSSVSYMD light chain CDR1 SEQ ID NO: 111 heavy cliain CDR3 STyyggdwyfnv SEQIDNO:112 light cliain CDR 1 RASSSVSYIH SEQ ID NO: 113 light chain CDR3 QQWTSNPPT SEQ ID NO: 114 heavy chain CDR1 GFTFNDYAMH SEQ ID NO: 115 heavy cliain CDR2 TISWNSGSIGYADSVKG SEQ ID NO: 116 heavy chain CDR3 DIQYGNYYYGMDV SEQ ID NO: 117 light chain CDR1 RASQSVSSYLA SEQ ID NO: 118 light chain CDR2 QASNRAT SEQ ID NO: 119 light cliain CDR3 QQRSNWPIT SEQ ID NO :120 heavy chain CDR 1 GYTFTSYNMH SEQ ID NO :121 heavy chain CDR3 VVYYSNSYWYFDV SEQ ID NO: 122 light chain CDR2 ■\PS\LAS SEQ ID NO: 123 light cliain CDR3 QQWSFNPPT SEQ ID NO :124 heavy chain CDR1 GYAFSYSW1N SEQ ID NO: 125 heavy chain CDR2 RIFPGDGDTDYNGKFKG SEQ ID NO :126 heavy cliain CDR3 NVFDGYWLVY SEQ ID NO: 127 light chain CDR 1 RSSKSLLHSNGITYLY SEQ ID NO :128 light chain CDR2 QMSNLVS SEQ ID NO: 129 light chain CDR3 AQNLELPYT SEQ ID NO: 130 heavy cliain ABR1 yrftnywih SEQ ID NO: 131 heavy cliain ABR2 WIGGINPGNNYATYRR SEQ ID NO: 132 heavy chain ABR3 TREGYGNYGAWFAY SEQ ID NO: 133 light chain ABR1 QSL AN S YGNTFL S SEQ ID NO: 134 light cliain ABR2 LL1YGISNRFS SEQ ID NO: 135 light chain ABR3 LQGTHQPY SEQ ID NO: 136 heavy chain ABR1 FAFSIYDMS SEQ ID NO: 137 heavy chain ABR2 WVAYISSGGGTTYY SEQ ID NO: 138 heavy cliain ABR3 RHSGYGTHWGVLFAY SEQ ID NO: 139 light chain ABR1 QDISWLA SEQ ID NO :140 light chain ABR2 LLIYYTSILHS SEQ ID NO: 141 light chain ABR3 QQGNTLPW SEQ ID NO :142 heavy cliain ABR1 YTFTSYWLHWO 2016/196344 PCT/US2016/034778 380 SEQ ID NO :143 heavy chain ABR2 WIGYINPRNDYTEY SEQ ID NO: 144 heavy chain ABR3 RRDITTFY SEQ ID NO: 145 light chain ABR1 QSVLYSANHKNYLA SEQ ID NO :146 light chain ABR2 LLIYWASTRES SEQ ID NO :147 light cham ABR3 HQYLSSW SEQ ID NO: 148 heavy chain ABR1 YEFSRSWMN SEQ ID NO :149 heavy chain ABR2 WVGRIYPGDGDTNYSGKF SEQ ID NO: 150 heavy chain ABR3 RDGSSWDWYFDV SEQ ID NO: 151 light chain ABR1 QSIVHSVGNTFLE SEQ ID NO: 152 light chain ABR2 LLIYKVSNRFS SEQ ID NO: 153 light chain ABR3 FQGSQFPY SEQ ID NO: 154 heavy chain CDR1 GYRFTNYWIH SEQ ID NO: 155 heavy chain CDR2 G1NPGNNYATYRRKFQG SEQ ID NO: 156 heavy chain CDR3 EGYGNYGAWFAY SEQ ID NO: 157 light chain CDR1 RSSQSLANSYGNTFLS SEQ ID NO: 158 light chain CDR2 G1SNRFS light chain CDR3 SEQ ID NO: 159 LQGTHQPYT SEQ ID NO :160 heavy chain CDR1 GFAFS1YDMS SEQ ID NO :161 heavy chain CDR2 YTSSGGGTTYYPDTVKG SEQ ID NO :162 heavy chain CDR3 HSGYGTHWGVLFAY light chain CDR1 SEQ ID NO: 163 RASQDISNYLA SEQ ID NO :164 light chain CDR2 YTSILHS SEQ ID NO :165 light chain CDR3 QQGNTLPWT SEQ ID NO: 166 gyietdyyit heavy chain CDR1 heavy chain CDR2 WIYPGSGNTKYNEKF SEQ ID NO: 167 SEQ ID NO :168 heavy chain CDR3 YGNYWFAY SEQ ID NO :169 light chain CDR 1 KASQSVDFDGDSYMN SEQ ID NO: 170 light chain CDR2 AASNLES SEQ ID NO: 171 light chain CDR3 QQSNEDPWT SEQ ID NO :172 heavy chain CDR1 YTFTTYWMH SEQ ID NO: 173 heavy chain CDR2 WIGYINPSTGYTDY SEQ ID NO: 174 heavy chain CDR3 TRRGPSY GNHGAWFP Y SEQ ID NO: 175 light chain CDR1 )3NVDTYVS SEQ ID NO :176 light chain CDR2 Lliygasnryt SEQ ID NO :177 light chain CDR3 GQSYRYPP SEQ ID NO: 178 heavy chain CDR1 GYTFTGYYTGH SEQ ID NO: 179 heavy chain CDR2 WIDPNSGATTYAQKF SEQ ID NO: 180 heavy chain CDR3 KTTQTTWGFPF SEQ ID NO: 181 light chain CDR1 RASQGVYQWLA SEQ ID NO: 182 light chain CDR2 KASHLYN SEQ ID NO: 183 light chain CDR3 QQLNSYPLT SEQ ID NO: 184 heavy chain CDR1 GYTFTDYWMH SEQ ID NO: 185 heavy chain CDR2 W1GYINPNTAYTDY SEQ ID NO: 186 light chain CDR1 KASENVDSFVS SEQ ID NO: 187 light chain CDR2 GASNRYT SEQ ID NO: 188 light chain CDR3 GQNYRYPLT SEQ ID NO: 189 heavy chain ABR1 FSL1SYGVH SEQ ID NO: 190 heavy chain ABR2 WLGVIWRGGSTDY SEQ ID NO: 191 heavy chain ABR3 kTLITTGYAMDY SEQ ID NO: 192 light chain ABR1 ED؛Y\H؛ A SEQ ID NO: 193 light chain ABR2 LLISGATSLETG SEQ ID NO: 194 light chain ABR3 QQYWSTP SEQ ID NO: 195 heavy chain ABR1 FTFNSFAMSWO 2016/196344 PCT/US2016/034778 381 SEQ ID NO: 196 heavy chain ABR2 WVSAISGSGGGTYY SEQ ID NO: 197 heavy chain ABR3 KDKTLWFGEPVFDY SEQ ID NO: 198 light cliain ABRI QSVSSYLA SEQ ID NO: 199 light cliain ABR2 LLIYDASNRAT SEQ ID N0:200 light chain ABR3 QQRSNWPP SEQ ID NO:201 heavy chain ABRI FSLTSYGVH SEQ ID NO:202 heavy chain ABR2 WIGVMWRGGSTDY SEQ ID NO:203 heavy chain ABR3 KSMITTGFVMDS SEQ ID NO:204 light chain ABRI ED1YNRLT SEQ ID NO:205 light chain ABR2 QLISGATSLET SEQ ID NO:206 light chain ABR3 QQYWSNPY SEQ ID NO:207 heavy chain ABR1 FDFSRSWMN SEQ ID NO:208 heavy chain ABR2 WIGEINPDSSTINY SEQ ID NO:209 heavy chain ABR3 RYGNWFPY SEQ ID NO:210 light chain ABRI QNVDTNVA SEQIDN0:211 light chai a ABR2 ALIYSASYRYS SEQ ID NO:212 light chain ABR3 QQYDSYPL SEQ ID NO:213 heavy chain ABRI GTFSSYAFS heavy cliain ABR2 SEQIDNO:214 WMGRVIPFLGIANS SEQ ID NO:215 heavy chain ABR3 RDDIAALGPFDY SEQ ID NO:216 light chain ABRI QGISSWLA SEQ ID NO:217 light cliain ABR2 SLIYAASSLQS SEQIDNO:218 light cliain ABR3 QQYNSYPR SEQIDNO:219 heavy chain ABRI YTFTDYWMQ SEQ ID NO:220 heavy chain ABR2 WIGTIYPGDGDTGY SEQIDNO:221 heavy cliain ABR3 XGDYYGSNSLDY SEQ ID NO:222 light chain ABR I QDVSTWA SEQ ID NO:223 light chain ABR2 RLIYSASYRY1 SEQ ID NO:224 light chain ABR3 QQHYSPPY SEQ ID NO:225 heavy cliain CDR1 GFSLTSYGVH SEQ ID NO :226 heavy chain CDR2 VMWRGGSTDYNAAFMS SEQ ID NO:227 heavy chain CDR3 SMITTGFVMDS SEQ ID NO:228 light chain CDR1 KASEDlYXRl L SEQ ID NO:229 light cliain CDR2 GATSLET QQYWSNPYT SEQ ID NO:230 light chain CDR3 SEQ ID NO:231 heavy chain CDR1 GFSLISYGVH SEQ ID NO:232 heavy cliain CDR2 VIWRGG STD YN AAFMS SEQ ID NO :23 3 heavy cliain CDR3 TLITTGYAMDY SEQ ID NO:234 light chain CDR1 KASEDIYNRLA SEQ ID NO:235 light chain CDR2 GATSLET SEQ ID NO:236 light cliain CDR3 QQYWSTPT SEQ ID NO:237 heavy cliain CDR1 GFDFSRSWMN SEQ ID NO:238 heavy chain CDR2 EINPDSSTINYTTSLKD SEQ ID NO:239 heavy chain CDR3 YGNWFPY SEQ ID NQ:240 light cliain CDR1 kASQNVDTNVA SEQ ID NO:241 light chain CDR2 SASYRYS SEQ ID NO:242 light chain CDR3 QQYDSYPLT SEQ ID NO:243 heavy chain ABRI FDFSRYWMS SEQ ID NO:244 heavy cliain ABR2 WIGELNPTSSTINF SEQ ID NO :245 heavy chain ABR3 RGXYYRYGDWDT SEQ ID NO:246 light chain ABRI KSVSTSGYSYLH SEQ ID NO:247 light chain ABR2 LLIYLASNLES SEQ ID NO:248 light cliain ABR3 QHSRELPFWO 2016/196344 PCT/US2016/034778 382 SEQ ID NO:249 heavy chain ABR1 STFTTYWIH SEQ ID NO:250 heavy chain ABR2 WIGYINPNTGYTEY SEQIDNO:251 heavy chain ABR3 VRFITVVGG SEQ ID NO:252 light chain ABR1 SSVSSSHLH SEQ ID NO:253 light chain ABR2 LWIYSTSNLAS SEQ ID NO:254 light chain ABR3 HQYHRSPL SEQ ID NO:255 heavy diain ABR1 FSLTTYGIGVG SEQ ID NO :256 heavy chain ABR2 WLTHIWWNDNKYY SEQ ID NO:257 heavy chain ABR3 YGYTY SEQ ID NO:258 light chain ABR1 QSLLYSNGNTYLH SEQ ID NO:259 light chain ABR2 LLIYKLSNRFS SEQ ID NO:260 light chain ABR3 SQSTHVPW SEQ ID NO:261 heavy chain ABR1 FNIKDTYIH SEQ ID NO:262 heavy chain ABR2 WVARIYPTNGYTRY SEQ ID NO:263 heavy chain ABR3 kWGGDGFYAMDY SEQ ID NO :264 light chain ABR1 QDVNTAVA SEQ ID NO:265 light chain ABR2 LLTYSASFLYS SEQ ID NO:266 QQHYTTPP light chain ABR3 SEQ ID NO :267 heavy diain ABR3 RWGGDGFYAMDV SEQ ID XO 268 YSFTSYWIA heavy chain ABR1 SEQ ID NO:269 heavy chain ABR2 YMGLTYPGDSDTKY SEQ ID NO:270 heavy diain ABR3 RHDVGYCSSSNCAKWPEYFQH SEQ ID NO:271 light chain ABR 1 SSNIGNNYVS SEQ ID NO:272 light chai 11 ABR2 LLIYGHTNRPA SEQ ID NO:273 light chain ABR3 AAWDDSLSGW SEQ ID NO:274 heavy diain ABR1 ypftnygmn SEQ ID NO:275 heavy chain ABR2 WMGWINTSTGESTF SEQ ID NO:276 heavy chain ABR3 RWEVYHGYVPY SEQ ID NO:277 light chain ABR1 QDVYNAVA.
SEQ ID NO:278 light diain ABR2 LLIYSASSRYT SEQ ID NO :279 light chain ABR3 QQHFRTPF SEQ ID NO:280 heavy chain ABR1 ITFSINTMG SEQ ID NO:281 heavy chain ABR2 LVALISSIGDTYYA SEQ ID NO:282 heavy diain ABR3 1KRFRTAAQGTDY SEQ ID NO:283 heavy chain CDR1 GFNIKDTYIH SEQ ID NO:284 heavy chain CDR2 RIYPTNGYTRYADSVKG SEQ ID NO:285 heavy diain CDR3 WGGDGFYAMDY SEQ ID NO :286 light chain CDR1 RASQDVNTAVA SEQ ID NO:287 light chain CDR2 SASFLYS SEQ ID NO:288 light chain CDR3 QQHYTTPPT SEQ ID NO:289 heavy diain CDR1 GFNIKDTYIH SEQ ID NO :290 heavy chain CDR2 RIYPTNGYTRYADSVKG SEQIDNO:291 heavy chain CDR3 WGGDGFYAMDY SEQ ID NO:292 light chain CDR1 )RASQDVNTAVA SEQ ID NO:293 light diain CDR2 SASFLYS SEQ ID NO :294 light chain CDR3 QQHYTTPPT SEQ ID NO:295 heavy chain CDR1 GYSFTSYWIA SEQ ID NO:296 heavy chain CDR2 LIYPGDSDTKYSPSFQG SEQ ID NO:297 heavy diain CDR3 HDVGYCSSSNCAKWPEYFQH SEQ ID NO :298 light chain CDR 1 SGSSSNIGNNYVS SEQ ID NO:299 light chain CDR2 GHTNRPA SEQ ID NO :300 light chain CDR3 AAWDDSLSGWV SEQ ID NO :301 heavy diain CDR1 gitfsintmgWO 2016/196344 PCT/US2016/034778 383 SEQ ID NO :302 heavy chain CDR2 LISSIGDTYYADSVKG SEQ ID NO :303 heavy chain CDR3 pRTAAQGTDY SEQ ID NO :304 heavy chain ABR1 FTFSDSWIH SEQ ID NO:305 heavy cliain ABR2 WVAWISPYGGSTYY SEQ ID NO :306 heavy chain ABR3 RRHWPGGFDY SEQ ID NO :307 light chain ABR1 QDVSTAVA SEQ ID NO:308 light chain ABR2 LLIYSASFLYS SEQ ID NO :309 light chain ABR3 QQYLYHPA SEQIDNO:310 heavy chain ABR1 YTfTSYVMH SEQ ID NOG 11 heavy chain ABR2 WIGYWTFNDGTKY SEQ ID NOG 12 heavy chain ABR3 RQAWGYP SEQ ID NO :313 light chain ABR1 ESVEYYGTSLVQ SEQIDNO:314 light chain ABR2 LLIYAASSVDS SEQ ID NOG 15 light chain ABR3 QQSRRVPY SEQ ID NOG 16 heavy chain ABR1 YTFTSYDVH SEQ ID NOG 17 WMGWLHADTGITKF heavy chain ABR2 SEQ ID NOG 18 heavy chain ABR3 RERIQLWFDY SEQ ID NOG 19 light chain ABR1 QGISSWLA SEQ ID NOG20 light chai n ABR2 SLIYAASSLQS SEQ ID NO :321 light chain ABR3 QQYNSYPY SEQ ID NOG22 heavy chain ABR1 DTFSTYAIS SEQ ID NOG23 WMGGHPH GkAHY heavy cliain ABR2 SEQ ID NOG24 heavy cliain ABR3 RKFHFVSGSPFGMDV SEQ ID NO :325 light chai 11 ABR1 QSVSSYLA SEQ ID NOG26 light chain ABR2 QLTYDASNRAT SEQ ID NO :327 light cliain ABR3 OQRSNWP SEQ ID NOG28 heavy chain ABR 1 FTFSSYIMM SEQ ID NOG29 heavy chain ABR2 WVSSIYPSGGITFY SEQ ID NOG30 heavy chain ABR3 RIKLGTVTTVDY SEQIDNOG31 light cliain ABR1 SSDVGGYNYVS SEQ ID NO:332 light chain ABR2 LMIYDVSNRPS SEQ ID NOG33 light chain ABR3 SSYTSSSTR SEQ ID NOG34 heavy chain CDR1 GFNTKDYTT..H SEQ ID NOG35 heavy cliain CDR2 WTNPDNGNTVYDPKFQG SEQ ID NOG36 heavy chain CDR3 RD YTYEKA/XLDY SEQ ID NOG37 light chain CDR1 RASGNIYNYLA SEQ ID NOG38 light cliain CDR2 DAKTLAD SEQ ID NOG39 light cliain CDR3 QHFWSLPFT SEQ ID NO :340 SET -1 A-combo27 KEFILRFSVAHKYVDSLNVIRSAIGTPLQTISSGGTSL LMID SG S GDNLF A VD VRGIDPEEGRFNNLRLI VERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS GDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO :341 SET -1 A-co mb028 KEF'l'LDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LMIDNIATMVATWDWGIDPEEGRFNNLRIJW.RN NLYVTGFVNRTNNVFYRF/XDFSHVTFPGTTAVILS GDSSYTTLQRVAGISRTGMQINRI-ISLTTSYLDLMSI-I SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINA1LGSVALILNCHHHASAVAA SEQ ID NO :342 SET -1A -combo29 KEFTLDFSTAKTYVDSLNVIRSAJGTPLQTISSGGTSL LMIDSNLVPMVATVDVRGIDPEEGRFNNLRLIVERNWO 2016/196344 PCT/US2016/034778 384 NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS GDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO:343 SET -1 A-combo3 0 KEF'IEDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LMIDSGILGFWn.DWGIDPEEGRFNNI,RLI\T;RNN LYWGFVNRTNNVFYRFADFSHWFPGTTAVTESG DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO :344 SLT-lA-combo31 KEF1LDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL LMTDSGSGDNLFAVGTLGFDFTLGRFNNLRLIVERN NIAAGTGFVNRTNNWYRFADFSHVTFPGTTAVTES GDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO:345 SLT-lA-combo32 KEFTLDFSTAKTYVDSLNV1RSA1GTPLQTISSGGTSL LMIDSGSGDNLFAVDILGFVFTLGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS GDSSYTTLQRVAGTSRTGMQINRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSWVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO :346 SET-1 A-combo3 3 KEF'FLDFSTAKTYVDSLNVIRSAIGl’PLQTISSGGTSL LMIDSGSGDNLFAVDIEGFDFTLGRFNNLRLIVERN NLYVTGFWRTNNWYRFADFSHVTFPGTTAVrFL׳S GDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRTSFGSINAILGSVALTLNCHHHASAVAA SEQ ID NO :347 SET -1 A-combo3 4 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LMID SG SGDNLFAVGILGFVFTLGRFNNLRLI VERN NLYVIGFVNRTNNVFYRF'ADFSHVTFPGTTAVTLS GDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO:348 KEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL SLT-lA-combo3 5 LMIDSGIGDNEFAVDVRGIAPIEARF'NNLRLIVERNN EYVTGFVNRTNNVFYRFADFSHVI'FPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYEDLMSHS ATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL AALSG AS Y VMTAED VDLTLNWGRLS S VLPD YI-IGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:349 SET -1 A-combo3 6 KEF1LDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LMIDSGSGDNLFAVGILGFVFTLEGRFNNLRLIVERN NI,Y\7TGF\7NRTNNWYRFADFSHVTFPGTTAVTI...S GDSSYTTLQRVAGISRTGMQINRHSLTrSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASAVAAWO 2016/196344 PCT/US2016/034778 385 SEQ ID NO:350 SLT"lA״combo37 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LMID SG SGDNLFAVNL VPMVATVGRFKNLRLIVER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV1L SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQIDNO:351 SLT-1 A-combo3 8 KEF'ILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LMIDSGSGDNI.,FAWWGIDPEEGRFNNI.,RLIVERN NLYVTGFVNRTNNVFYRF/XDFSHVTFPGTNLVPMV atvsyttlqrvagisrtgmqinrhslttsyldlmsh SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO:352 SET -1A -combo3 9 KEFTLDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL LMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERN NTAA؟GFVWTNNWYRFADFSHVTFPGTTAVTI...S GDSSYTTLQRVAGISRTGMQINRHSLTTSY LDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRGI LGDVFTLSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:353 SLT-lA-combo40 KEF'rtDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLTVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS GDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSWVGRISFGSINAILGSVALILNCHHHILRFSVA HKASAVAA SEQ ID NO:354 SLT-1 A-combo41 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LMIDSGSGDNLFAVDILGFDFTLGRFNNLRLIVERN NLY\'TGFV7NRTNNWYRFADFSHVTFPGTTAX,TI...S GDSSYTTLQRVAGISRTGMQTNRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHARNLVP MVATVASAVAA SEQ ID NO:355 SLT-lA-basel KEF1LRFSVAHKYVDSLNVIRSAIGTPLQTISSGGTSL LMID SGSGDNLFAVD WGIDPEEGRFNNLRLI VERN NLYVT'GFVNRTNNVFYRF'ADFSHVTFPGTTAVTLS GDSSYTTLQRVAGTSRTGMQINRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASRVAR SEQ ID NO:356 SLT-lA-base2 and KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LMIDNLVPMVATVVDVRGIDPEEGRFNNL.RLIVERN StxA-basel NLYVTGFYTxiRTNNWYRFADFSHVTFPGTTAV'FL.S GDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASRVAR SEQ ID NO:357 SLT-lA-base3 and KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL S؛xA-base2 LMIDSNLVPMVATVDVRGIDPEEGRFNNLRLIVERN ?JLYVrGFVNRTNNVFYRFADFSHVTFPGTTAVTLS GDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHVI VI VI VI VI VI VI a i VI 00 % 0) TO Q Q C3 ^p ^p ^p ^p UC VD <* <* p—1 5^ H H H ^5 J J o GO go v/j GO GO GO GO GO v/j GO CO O־- CN V*) o *- m ‘/ו *•v co f-r• f-r• C'־) m co o O O b b o O o z z z z z z O o O O' O o o O Hi W a a w V) GO ■/) GO GO GO WO 2016/196344 PCT/US2016/034778 386 SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASRVAR ID ase4 and KEFTLDFSTAKTYVDSLNVIR SAIGTPLQTISSGGTSL LMID SGILGF WH.D WGIDPE EGRFXXLRMVEHXX' LYWGFVNRTNNWWFADF: SHVTFPGTTAVTLSG DSSYTTLQRVAGISRTGMQIN RHSLTTSYLDLMSHS GTSLTQSVARAM..RFVTVTA:E ALRFRQ1QRGFR11L DDLSGRSYVMTAEDVDLTLNA VGRLS S VLPDYHGQ JLNCHHHASRVAR DSVRVGRISFGSINAILGSVAL ase5 KEFTLDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL LMTDSGSGDNLFAVGTLGFDFTLGRFNNLRLIVERN NIAA^TGFVNRTNdXWYRFADFSHVTFPGTTAVTLS GDSSYTTLQRVAGISRTGMQTNRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASRVAR ID ase6 KEFTLDFSTAKTYVDSLNV1RSA1GTPLQTISSGGTSL LMIDSGSGDNLFAVDILGFVFTLGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS GDSSYTTLQRVAGTSRTGMQINRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINATLGSVALILNCHHHASRVAR ID KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LMIDSGSGDNLFAVDILGFDFTLGRF’NNLRLIVERN NLYVTGF\rNRTNNV'FYRFADFSHVTFPGTTAVrFL.S GDSSYTTLQRVAG1SRTGMQINRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASRVAR ID KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LMIDSGSGDNLFAVGILGFVFTLGRFNNLRLIVERN NLYVTGFVNRTNNVFYRF'ADFSHVTFPGTTAVTLS GDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASRVAR ID ase9 and KEFILDFSTAKTYVDSLNVIRSAIGTPLQnSSGGTSL IA4IDSGIGDNI,FAVDWGIAPIEARFNNI.RLIW,RNN LYVTGFVNRTNNVFYRFADFSHVI'FPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS ATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL AALSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASRVAR ID iselO KEFTLDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL LMTDSGSGDNLFAVGTLGFVFTLEGRFNNLRLIVERN NTYWGFVWTNNWYRFADFSI-IVTFPGTTAVTI...S GDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASRVAR ID !sell KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LMIDSGSGDNLFAVNLVPMVATVGRFNNLRLIVER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLWO 2016/196344 PCT/US2016/034778 387 SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS HSGTSLTQSVARAMLRFVIVLAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASRVAR SEQ ID NO :366 SET-!Abase 12 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL IA4IDSGSGDNLFA\T)WGIDPEEGRFNNLREIWJ3.N NLYVTGFVNRTNNVFYRFADFSHVTFPGTNLVPMV ATVSYTTLQRVAGISRTGMQINRHSLTTSYI,DEMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASRVAR SEQ ID NO :367 SLT-lAbasel3 KEFTLDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL LMTDSGSGDNLFAVDVRGTDPEEGRFNNLRLIVERN Nl.A'VTGFVNRTNN VFYRFADFSH VTFPGTTA VTL.S GDSSYTTLQRVAGISRTGMQTNRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRGI LGDVFTLSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNSHHHASRVAR SEQ ID NO:368 SET-!Abase 14 KEFTLDFSTAKTYVDSLNV1RSA1GTPLQTISSGGTSL LM1DSGSGDNLFAVDVRG1DPEEGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS GDSSYTTLQRVAGTSRTGMQINRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHILRFSVA HKASRVAR SEQ ID NO:369 SEI’-!Abase 15 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LMIDSGSGDNLFAVDILGFDFTLGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS GDSSYTTLQRVAG1SRTGMQINRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHARNLVP MVATVASRVAR SEQ ID NO :3 70 Shiga Toxin A KEFILRFSVAHKYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGIGDNLFAVDVRGIDPEEGRFNNLRLIVERN Polypeptide NLYVTGFVNATNNAFYRFADFSHV^PGTTAVTLS combo 101 ADSSYTTLQRVAGISRTGMQ1NRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO :371 Shiga Toxin A AEFILRFSVA1-IKYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGIGDNEFAVDVRGIDPEEGRF’NNLRLIVERN Polypeptide NLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLS combo 102 ADSSYTTLQRVAGISRTGMQINRI-ISLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:372 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMTONLWMVATVVDVRGIDPEEGRFNNLRLIVERN Polypeptide NLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLS combo 103 ADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNSHHHASAVAAWO 2016/196344 PCT/US2016/034778 388 SEQ ID NO :3 73 Shiga Toxin A KEFILRFSVAHKYVDSLNVIRSAIGTPLQTISSGGTSL LMIDSGIGDNLFAVDVRGIDPEEGRFNNLRLIVERN Subunit Effector Polypeptide NLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLS combo 104 ADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVALTLNWGRLSSVLPDYHG QDSVRVGR1SFGSINAILGSVALILNSHHHASAVAA SEQ ID NO :374 Shiga Toxin A KEF'l'LDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSNIA7PMVAT\7D\TtGIDPEEGRFNNT..RLI\/ERN Polypeptide NLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLS combo 105 ADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSH SGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO :375 Shiga Toxin A KEFTLDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL Subunit Effector LMIDSNLVPMVATVDVRGIDPEEGRFNNLRLIVERN Polypeptide NLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLS ADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSH combo 106 SGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVDLTLNWGALSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO :376 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGILGFVFTLDVRGIDPEEGRFNNLRLIVERNN Polypeptide LYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo 107 DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSLTQSVARAM..RFVTVTAEALRFRQIQRGFRTTI, DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGR1SFGSINAILGSVALILNSHHHASAVAA SEQ ID NO :377 Shiga Toxin A KEFT'LDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGILGFVFTLDVRGIDPEEGRFNNLRLIVERNN Polypeptide LYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo 108 DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRTSFGSINAILGSVALTLNSHHHASAVAA SEQ ID NO :3 78 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGIGDNLFAVDILGFDFTLGRFNNLRL1VERNN Polypeptide LYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo 109 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:379 Shiga Toxin A KEFILDFITAKTYVDSLNVIRSAIGTPLQTISSGGTSLL Subunit Effector MIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNL Polypeptide YVTGFVNRTNNM^RF/IDFSHVTFPGTTAVTLS/IDS combo 110 SYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGT SLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDD LSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDS VRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:380 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LNnDSGIGDNIEAVGILGFDFTI..GRFNNT..RLI\/ERNN Subunit Effector Polypeptide LYVrGFVNATNN.'YFY'RF/XDFSHVTFPGTTAVTLSA combolll DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLWO 2016/196344 PCT/US2016/034778 389 DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHI-IHASAVAA SEQ ID NO:381 Shiga Toxin A MEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISIGGTSL Subunit Effector LM1DSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNN Polypeptide LYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo 112 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSVARAM..RFVTVTAEALRFRQIQRGFRTTI, DDLSGASYA'7MTAEDWLT[,NWGRLSSXT.,PDYT-IGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:382 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMTDSGIGDNLFAVGILGFDFTLGRFNNLRLTVERNN Polypeptide L.YVTGFVNATNNVFYRFADFSHVTFPGTTAVTIAA combo 113 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSV’RVGRISFGSINAILGSVALILNCHHHASAVAzX SEQ ID NO:383 Shiga Toxin A KEFTLDFSTAKTYVDSLNV1RSA1GTPLQTISSGGTSL Subunit Effector LMIDSGIGDNLFAVGILGFDFTLGRFNNLRL1VERNN Polypeptide LYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo 114 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINA1LGSVALILNSHHHASAVAA SEQ ID NO:384 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIVERNN Polypeptide LYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo 115 DSSYTTLqRVAG1SRTGMqINRHSLTTSYLDLMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:385 Shiga Toxin A AEFTLDFITAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LM1D SGIGDNLF AVDILGFVFTLGRFNNLRLIVERNN Polypeptide LYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo 116 DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVAL1LNCHHHASAVAA SEQ ID NO:386 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector IA4ID SGIGDNI.F A VT)ILGFDFTLGRFNW,RLIYTSRNN Polypeptide LYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo 117 DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:387 Shiga Toxin A KEFTLDFSTAATYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMTDSGIGDNLFAVDILGFDFTLGRFNNLRLTVERNN Polypeptide L.YVTGFVNATNNVFYRFADFSHVTFPGTTAVTIAA combo 118 DSSY؟TLQRVAGISRTGMQINRHSLTTSYI>DLMSHS GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:388 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIWRNN Polypeptide LYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo 119 DSSYTTLQRVAGISRTGMQINRHSLTTSYLAI.MSHSWO 2016/196344 PCT/US2016/034778 390 GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:389 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector IA4ID SGIGDNI.F AWILGF WTLGRFNW.RLIATSRNN Polypeptide LYWGFVNRTNNAFWFADFSHWFPGTTAWLSA combo 120 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS ATSLTQSVARAM..RFVTVTAEALRFRQIQRGFRTTI, DDLSGASYWTAEDWLTL,NWGRLSSXT,PDTO DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:390 Shiga Toxin A KEFTLDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL Subunit Effector LMTDSGIGDNLFAVGILGFDFTLGRFNNLRLTVERNN LYVTGFVNRTNNAFYRFADFSH VTFPGTTAVTLSA Polypeptide combo 121 DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQIDNO:391 Shiga Toxin A KEFTLDFSTAKTYVDSLNV1RSA1GTPLQTISSGGTSL LMIDSGIGDNAFAVGILGFDFTLGRFNNLRLIVERNN Subunit Effector Polypeptide LYVTGFVNRTNMAFYRFADFSHVTFPGTTAVTLSA combo 122 DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:392 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN Polypeptide LYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo 123 DSSYTTLqRVAG1SRTGMqINRHSLTTSYLDLMSHS gtsltqsvaramlrfvtvtaealrfrqiqrgfrttl ddlsgasyvmtaedvaltlnwgrlssvlpdyhgq DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:393 Shiga Toxin A keftldfstaktyvdslnvirsaigtplqtissggtsl LMIDSGIGDNLTAVDILGFDFTLGRFNNLRLIVERNN Subunit Effector Polypeptide LYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo 124 DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGR1SFGSINAILGSVAL1LNSHHHASAVAA SEQ ID NO:394 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector IA4ID SGIGDNI.F AVGILGFDFTLGRFNNI.RLIYTSRNN LYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA Polypeptide combo 125 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:395 Shiga Toxin A KEFTLDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL Subunit Effector LMTDSGIGDNLFVVGILGFDFTLGRFNNLRLTVERNN Polypeptide LYXffGFV’NRTNNYTYRFADFSHVTFPGTTAY'TLSz'k combo 126 DSSY؟TLQRVAGISRTGMQINRHSLTTSYI>ALMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:396 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGIGDNLFAVGILGVDFTLGRFNNLRLIVERNN LYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSAWO 2016/196344 PCT/US2016/034778 391 Polypeptide DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS combo 127 GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHI-IHASAVAA SEQ ID NO:397 Shiga Toxin A MEF1LDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS Subunit Effector LLMIDSGIGDNI,FAVGILG\T)FTI,GRFNNI,RLIAT;RN Polypeptide NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS combo 128 ADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVALTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:398 Shiga Toxin A KEFTLDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL Subunit Effector LMTDSGIGDNLFAVDILGFDFTLEGRFNNLRLIVERN Polypeptide Ni.A'VTGFVNRTNN VFYRFADFSH VTFPGTTA VTL.S combo 129 ADSSYTTLQRVAGISRTGMQTNRHSLTTSYLDLMSH SGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVALTLNWGRLSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:399 Shiga Toxin A KEFTLDFSTAKTYVDSLNV1RSA1GTPLQTISSGGTSL Subunit Effector LM1DSGIGDNLFA VDILGFDFTLEGRFNNLRLI VERN Polypeptide NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS combol30 ADSSYTTLQRVAGTSRTGMQINRHSLTTSYLDLMSH SGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVALTLNWGRLSSWPDYHG QDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID N0:400 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGIGDNLFAVG1LGFVFTLEGRFNNLRLIVERN Polypeptide NLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLS combo 131 ADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHG QDSVRVGRTSFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:401 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGIGDNLFAVGILGFVFTLEGRFNNLRLIVERN Polypeptide NLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLS combo!32 ADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVALTLNWGRLSSVLPDYHG QDSVRVGR1SFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:402 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN Subunit Effector Polypeptide LYVTGFVNATNNAT'YRF'ADFSHVTFPGTTAVTLSA combo 133 DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:403 Shiga Toxin A AEFTLDFIAAKTYVDSLNVTRSATGTPLQTISSGGTSL Subunit Effector LAnDSGIGDNIEA\TJILGFDFTT.GRFNNT..RLI\/ERNN Polypeptide LYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo 134 DSSYTTLQRVAGISRTGMQINRHSLTTSYI.DLMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHI-IHASAVAA SEQ ID NO:404 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMID SGIGDNLF A VDILGFVFTLGRFNNLRLIVERNNWO 2016/196344 PCT/US2016/034778 392 Polypeptide LYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo 135 DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGR1SFGSINAILGSVAL1LNSHHHASAVAA SEQ ID NO:405 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSL Subunit Effector LMID SGIGDNI.F A WILGF WTLGRFNW.RLIATSRNN Polypeptide lAAHXiFVNATNNAFYRFADFSHVTFPGTTAVTLSA combol36 DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:406 Shiga Toxin A KEFTLDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL Subunit Effector LMTDSGIGDNLFAVGILGFVFTLGRFNNLRLTVERNN LYVTGF vNATNNVFYRFADFSHVTFPGTTAVTlAA Polypeptide combo 137 DSSYHTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:407 Shiga Toxin A MEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGIGDNLFAVGILGFVFTLGRFNNLRLIVERNN Polypeptide LYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo 138 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO:408 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN Polypeptide LYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo 139 DSSYTTLqRVAG1SRTGMqINRHSLTTSYLALMSHS GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRTSFGSINAILGSVALTLNSHHHASAVAA SEQ ID NO:409 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN Subunit Effector Polypeptide LYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo 140 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGALSSVLPDYHGQ DSVRVGR1SFGSINAILGSVAL1LNSHHHASAVAA SEQ ID NO:410 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGIGDNLFAVDILGFVFTLGRF'NNLRLIVERNN Polypeptide LYVTGFVNATNNVFYRF'ADFSHVTFPGTTAVTLSA combo 141 DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS gtsltqsaaramlrfvtvtaealrfrqiqrgfritl DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NOAH Shiga Toxin A AEFTLDFSTAKTYVDSLNVIRSAJGTPLQTISCGGTSL LMIDSGIGDMEA\TJILGFVFTI>GRFNNI..RI.,I\׳ERNN Subunit Effector Polypeptide LYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo 142 DSSY^TTLQRVAGISRTGMQINRHSLTTSYI.DLMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA9ifon a i Gas sigon a i Oas WO 2016/196344 PCT/US2016/034778 393 SEQ ID NO:412Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNN Subunit Effector Polypeptide LYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo 143 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGR1SFGSINAILGSVAL1LNSHHHASAVAA SEQ ID NO:413Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGIGDNATAVGILGFDFTLGRFNM.RLIW.RNN Polypeptide LYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo 144 DSSYTTLQRVAGISRTGMQINRI-ISLTTSYLALMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQIDNO:414 Shiga Toxin A KEFTLDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL LAnDSGIGDM.EAVGILGFDm.GRFNNT..RLI\/ERNN Subunit Effector Polypeptide LYWGFV’NRTNNAFYRFADFSHVTFPGTTAWLSzX combo 145 DSSYTTLQRVAGISRTGMQINRFISLTTSYLALMSHS GTSLTQSVARAMLRFVTVTAEALRFRQ1QRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA Shiga Toxin A KEFTLDFIIAKTYVDSLNVTRSAIGTPLQTISCGGTSL Subunit Effector LMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNN Polypeptide LYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo 146 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GlSLlQSVARAMLRr VIV1AEALRFRQ1QRGFR1TL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGR1SFGSINAILGSVALILNSHHHASAVAA Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNN Polypeptide LYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo 147 DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRTSFGSINAILGSVALTLNSHHHASAVAA SEQ ID NO:417Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSA1GTPLQTISCGGTSL Subunit Effector LMIDSGIGDNLFAVGILGFDFTLGRFNNLRL1VERNN Polypeptide LYVTGFVNRTNMAFYRFADFSHVTFPGTTAVTLSA combo 148 DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINATLGSVALILNSHHHASAVAA SEQ ID NO:418 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subimit Effector LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN Polypeptide LYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA combo 149 DSSYTTLQRVAGISRTGMQINRI-ISLTTSYLALMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQIDNO:419Shiga Toxin A AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LAnDSGIGDM.EA\TJILGFDm.GRFNNT..RLI\/ERNN Subunit Effector Polypeptide LYVTGFVNATNN.'XFY'RF/XDFSHVTFPGTTAVTLSA combo 150 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTLWO 2016/196344 PCT/US2016/034778 394 DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHI-IHASAVAA SEQ ID NO:420 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN Polypeptide LYWGFVNATNNAFWFADFSHWFPGTTAWLSA combo 151 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSAARAM..RFVTVTAEALRFRQIQRGFRTTI, DDLSGASYYAfTAEDXTDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:421 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMTDSGIGDNLFAVDILGFDFTLGRFNNLRLTVERNN Polypeptide L.YVTGFVNATNNAFYRFADFSHVTFPGTTAVTIAA combo 152 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSV’RVGRISFGSINAILGSVALILNCHHHASAVAzX SEQ ID NO:422 Shiga Toxin A KEFTLDFSTAKTYVDSLNV1RSA1GTPLQTISSGGTSL Subunit Effector LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN Polypeptide LYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combol53 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINA1LGSVALILNSHHHASAVAA SEQ ID NO:423 Shiga Toxin A MEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS Subunit Effector LLMTOSGIGDNLFAVDILGFDFTLGRFNNLRLIVERN Polypeptide NLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLS combo 154 ADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVALTLNWGRLSSVLPDYHG QDSVRVGRISFGSINA1LGSVALILNCHHHASAVAA SEQ ID NO:424 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN Polypeptide LYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combol55 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGR1SFGSINAILGSVAL1LNSHHHASAVAA SEQ ID NO:425 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector IA4ID SGIGDNI.F A VT)ILGFDFTLGRFNW,RLIYTSRNN Polypeptide LYYffGFVNATNNAFY'RFADFSHY'TFPGTTAXffLSA combo 156 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:426 Shiga Toxin A KEFTLDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL Subunit Effector LMTDSGIGDNLFAVDILGFDFTLGRFNNLRLTVERNN Polypeptide L.YVTGFVrxATNNAFYRFADFSHVTFPGTTAVTIAA combo 157 DSSY؟TLQRVAGISRTGMQINRHSLTTSYI>DLMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:427 Shiga Toxin A AEFILDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSL Subunit Effector LKffDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN Polypeptide LYVTGFVNATNNAFYTIFADFSHVTFPGTTAVTLSA combo!58 DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSWO 2016/196344 PCT/US2016/034778 395 GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHI-IHASAVAA SEQ ID NO:428 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector IA4IDSGIGDNIJ؟\VT)ILGFDFTLGRFNW^ Polypeptide LYWGFVNRTNNAFWFADFSHWFPGTTAWLSA combol59 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSAARAM..RFVTVTAEALRFRQIQRGFRTTI, DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:429 Shiga Toxin A KEFTLDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL Subunit Effector LMTDSGIGDNLFAVDILGFDFTLGRFNNLRLTVERNN LYVTGFVNRTNNAFYRFADFSH VTFPGTTAVTLSA Polypeptide combo 160 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGALSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:430 Shiga Toxin A KEFTLDFSTAKTYVDSLNV1RSA1GTPLQTISSGGTSL Subunit Effector LM1DSGIGDNLFAVDILGFDFTLGRFNNLRL1VERNN Polypeptide LYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo 161 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQIDNO:431 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN Polypeptide LYVTGFVNRTNNAFYRFADFSHVTFPGTNLVPMVA combo 162 TVSYTTLQRVAGISRTGMQ1NRHSLTTSYLDLMSHS GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:432 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN Subunit Effector Polypeptide LYVTGFVNRTNNAFYRFADFSHVTFPGTNLVPMVA combo 163 TVSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEAVALTLNWGRLSSVLPDYHGQ DSVRVGR1SFGSINAILGSVAL1LNSHHHASAVAA SEQ ID NO:433 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector IA4IDSGIGDNIJG\VT)ILGFDFTLGRFNW^ LYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA Polypeptide combo 164 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRGIL GDVFTLSYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:434 Shiga Toxin A AEFILDFSIAKWVDSLNMRSAIGTPLQTISSGGTSLL Subunit Effector MIDSGIGDNLFAVDILGFDFTLGRFNNLRLI\/ERNNL Polypeptide YAG'GFY'NRTNNYTYRFADFSHYffFPGTTAWLSADS SYTTI...QRVAGISRTGMQINRHSLTTSYTALMSHSGT combo 165 SLTQSAARAMLRFVTVTAEALRFRQIQRGFRGILGD VFTLSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSV RVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO:435 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LKffDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN LYVTGFVNATNNAFYTIFADFSHVTFPGTTAVTLSAWO 2016/196344 PCT/US2016/034778 396 Polypeptide DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS combo 166 GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHILRFSVAHK ASAVAA SEQ ID NO:436 Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit Effector LMIDSGIGDbMAWILGFDFTLGRFNM,RLI^ Polypeptide LYAT'GFVNATNNAFWFADFSHWFPGTTAWLSA combo 167 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGALSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHILRFSVAHK ASAVAA SEQ ID NO:437 Shiga Toxin A KEFTLDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL Subunit Effector LMTDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN Polypeptide LYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo 168 DSSY^TTLQRVAGISRTGMQINRHSLTTSYI.ALMSHS GTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSV’RVGRISFGSINAILGSVALILNSHHHARNI.,VPMV ATVASAVAA SEQ ID NO:438 Shiga Toxin A MEFTLDFIIAKTYVDSLNVIRSAIGTPLQTISCGGTSL Subunit Effector LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN Polypeptide LYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo 169 DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHS GTSLTQSAARAM..RFVTVTAEALRFRQIQRGFRTTI, DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHARNLVPMV ATVASAVAA SEQ ID NO:439 Cell-targeting KEFILDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL molecule 1 LMTDSGIGDNLFAVDILGFDFTLGPJ-'NNLRLTVERNN LYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSG DSSYTTLQRVAGISRTGMQINRHSLTTSYI.DI.MSHS GTST..TQS V AR AMLRFVTVT AE ALRFRQIQR GFRTTL DDLSGRSYVMTAEDVDLTI/NWGRLSSVLPDYHGQ DSVRVGRTSFGSINAIIXiSVAIJL.NSHHHASAYAAEF PKPSTPPGSSGGAPDIQMTQSPSST.GASVGDRVTITC K.ASEDIYNRLTW^rQQKPGkAPKLLISGATSLETGW SRFSGSGSGTDFTFTISSI vQPEDIATA^CQQYWSNP’Y TFGQGTKVEIKGSTSGSGK.PGSGEGSTKGQVQLQES GPGLVRPSQILSLTCTVSGFSLTSYGVHWVRQPPGR GLE WIG VM WRGG STD Y N A?d7MSP±Nr1'KDNSKNQ VSLRLSSVTzXADIAVYYCzXKSAnTIGFVWSWGQ G SF VIV S S SEQ ID NO:440 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 2 LMIDSGIGDNI..FAVDIT..GFDFTL.GRFNNI,RIJYTJtKM i v\׳:(a-\ VR : V:<: ADiGiA n-'PG: r.WG SG DSSYrTTI.QP..VAGISRTGMQIMtI-ISLTTSAT..DL.MSHS GTSLTQSVARAM..RFVTVTAEALRFRQIQRGFRTTI..
DDLSGRSYVMTAEDVDLTT.NWGRLSSX'I.PDYHGQ DSVRVGRISFGSINPJLGSVzXLILNSl-IHHASAVAAEF Pf^STPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC KASQDVNTAVAWYQQKPGKAPKLL1YSASFLYSGV PSRF'SGSRSGTDFTLl'lSSLQPEDFATYYCQQHYTTP prFGQGIKVElKGGGGSGGGGSGGGGSGGGGSGGGWO 2016/196344 PCT/US2016/034778 397 GSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYI HWVRQAPGKGLEWVARIYPTNGYTPA’ADSVKGRF TISADTSKNTAYLQMNSLPAEDTAVYYCSRWGGD GFYzAMDYWGQGTLVTVSS SEQ ID NO:441 Cell-targeting KEF'l'LDFSTAKTYVDSLNVIRSAIGm.QTISSGGTSL molecule 3 ; XRilSGSGDXLFAVXLYFXA A3 YGnlXX: J<1 : R XXiY X TGiA XL rXXS'F3 :>:■ \i): SiAIPPGil AV1L SGDSSAYTLQRVAGTSRTGMQINRHSLTTSYLDLMS riSGTSLTQSVARAMJtFVTVTAEALRFRQTQRGFRT TLDDLSGRSYVMT.XEDVDLTLNWGBDSSVLPDYHG QDSVRVGRISFGSINAILGSVALILNCHHPIASAVAAE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNP Y1TGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQE SGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPG RGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKN QVSLRLSSVTAADTAVYYCAKSMHTGFVMDSWG QGSLVTVSSA SEQ ID NO:442 Cell-targeting KEFTLDFSTAKTYVDSLNXTRSATGTPLQTISSGGTSL molecule 4 LMIDNLVPMVATVVDVRGIDPEEGRENNLRLIVERN NLYVTGFVNRTNNX'FYRFADFSHVTFPGTTAVILS GDSSYTTLQRVAGISRTGMQlNPJlSLTTSYLDLMSI-1 SGTSLTQSVARAMLRFVrVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG QD S VR VGRISFG SIN ATLG S VALILN CHHH A S A V A AE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CKASED1YNRLTWYQQKPGKAPKLL1SGATSLETGV PSRFSGSGSGTDFTFTISSLQPEDIArYYCQQYWSNP YTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQE SGPGT.A'TtPSQTI.SLTCTVSGFSLTSYGVHWWQPPG RGLEWIGVMWRCKjSTDYNAAFMSRLNITKDNSKN QVST..R.LSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSSA SEQ ID NO:443 Cell-targeting QVQLQQPGAJSLVKPGASVKMSCRI'SGYTFTSYNVM molecule 5 WVKQ’TPGQGLEWIGAIYPGNGDTSFNQKFKGKATL TADKSSST^/YMQESSETSEDSAVYYCARSN YY'GSS yxavffdvwgagttvtvssgstsgsgkpgsgegsqi VLSQSPT1LSASPGEKVTMTCRASSSVSYMDWYQQ KPGSSPKPWTYATSNI.ASGVPARFSGSGSGTSYSLTT SRVEAEDAA,n,YCQ(jWISNPPTFGAGTKLELKEFPK PSTPPGSSGGAPKEFTLDFSTAKTYY'DSLNVIRSAIG TPI.QTISSGGTSLLMIDSGTGDNLFAVDIT..GFDFTT.,GR FNNLRUVERYNIYVTGFVYRTNNyFYRFADFSHVT FPGTTAVTLSADSSYTTLQRVAGISRTGMQTYRHSL TTSYT.,DLMSHSGTSLTQSVARzAMI.,RF\,T\,TzAEzALR FRQIQRGFRTTL.DDLSGASYAAITAEDWT..TIJNWGR LSSX4..PD’n-IGQDS\׳IlVGRISFGSINAIIX؛SVALTLNC PII-THASRVAR SEQ ID NO:444 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSE molecule 6 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRIT'v'ERNN LYYYGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA D S S YTTLQRVAGISRT GMQINRH SETTS YLDLM SH S GTSLTQSVARAMT..RFVTVTAEALRFRQIQRGFRTTL DDI..SGASAAAITAEDW4LT1..NWGRI..SSXT.PDAT-IGQWO 2016/196344 PCT/US2016/034778 398 DSVRVGRISFGSINAILGSVALILNCHHHASAVAAEF PKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMS CKTSGYTFTSYNVHWVKQIPGQGLEWIGAIYPGNG DTSFNQKFKGKATLTzWKSSSTVYMQLSSLTSEDSA VYYCARSNTYYGSSYVWFFDVWGAGTr\7TVSSGST SGSGKPGSGEGSQIVLSQSPT1LSASPGEKVTMTCRA SSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPAR FSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTF GAGTKLELK SEQ ID NO:445 Cell-targeting QVQLQQPGAELVKPGASVKMSCKTSGYIFrSYNVH molecule 7 WVKQTPGQGLEWIGAIYPGNGDTSFNQKF'KGKATL TADKSSSTVYMQLSSLTSEDSAVYYCzikRSNYYGSS YVWFFDVWGAG'ITVTVSSGSTSGSGKPGSGEGSQI VLSQSPTILSASPGEKVTMTCRASSSVSY'MDWYQQ KPGSSPKPWIYATS'NLASGVPARFSGSGSGTSYSLTI SRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPK PS'FPPGSSGGAPKEFY'LDFSTAKTY VDSLNVIRSAIG ■PPLQTISSGGTSLLMIDSGTGDNLFAVDILGFDFTLGR FNNLRLIVERNNLYV'lXiFVNRTNNVFYREADFSHVT FPGTTAVTLSADSSYTTLQRVAG1SRTGMQTYRHSL TTSYI.DL.MSHSATSLTQSVARAMI.RFVTVTAEALR FRQIQRGFRTTLDDLSGASYVMTAEDV7DLTIJ?4WGR LSS'VLPDYHGQDSVRVGRTSFGSINAII,GSVALTLNC HHHASRVAR SEQ ID NO:446 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 8 LMIDSGIGDNLFAVDILGFDFTEGRFNNLRLIVERNN L¥YrrGFVNRTN־NVFYRFADFSHVTFPGTTAVTLSA DSSYTFLQRVAGISRl’GMQINRHSLTTSYLDLMSHS ATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTI.NWGRLSS\,I.PDYHGQ DSVRVGRISFGSTNAILGSVAL1I.NCHHHASAVAAEF PKPSTPPGSSGGAPQVQI.QQPGAELVKPGASVKMS CKTSGYTFTSYNVHVvVKQTPGQGLEWTGAIYPGNG DTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDSA VYYTARSNYYrGSSY\YvTFDVWGAGTT\7TVSSGST SGSGKPGSGEGSQIVI،SQSPTIT..SASPGEKVTMTCRA SSSVSAA4D"AYrQQKPGSSPKPWIYATSM.ASGWAR FSGSGSGTSYSI.YISRWAEDAATYY'CQQWTSNPPTF GAGI'KLELK SEQ ID NO:447 Cell-targeting KEFrL..DFSTAKTYVDST..NVTRSATGTPLQTISSGGTSL molecule 9 LYnDSGIGDNIFAVDILGFDFTL,GRF?GSlT..RlJVERNN [.YVTGFVNRTNNWYRFADFSHVTFPGTTAYTLSA DSSYTTLQRX'AGISRTGMQINRHSLTTSYI.DI.MSHS GTST..TQS V AR AMLRFVTVT AE ALRFRQIQR GFRTTL DDLSGASYVMTAEDY,DT..TLNWGRLSSVLPDYHGQ DSVRVGRTSFGSINAII.GSVAIJL.NSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSST..SASVGDRVTITC RASQGISSWI,AWYQQKPEKAPK.SLTYAASSLQSGVP SRFSGSGSGTDFTIvTTSSLQPEDFATYYCQQYNSYPR TP GQG IRVEIKGGGGSQVQL VQSG/W VKKPGSS VR VSCKASGGTFSSYAFSWVRQ/APGQGLEWMGRVIPF LGIANSAQKFQGRVTITADKSTSI'AYMDLSSLRSED TAVYYCzXRDDIAzALGPFDYWGQGTLVI'VSS SEQ ID NO:448 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 10 LMIDSGIGDNI,FAVDn..GFDFII,GRFNFn,RIJVERNNWO 2016/196344 PCT/US2016/034778 399 LYVrGFVNRrFTNVFYRIGADFSHVTFPGTTAVTLSzA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSLTQSVARAMLRFVTVGAEALRFRQIQRGFRCT^ DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGR1SFGSINAILGSVAL1LNSHHHASAVAAEF PKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATTSC RATESVEYYGTSLVQWYQQKPGQPPKLLTYAASSV DSGVPARFSGSGSGTDFSLT1HPVEEDDLAMYFCQQS RRVTYTFGGGTKLETKGGGGSEVQI.QQSGPELVKPG ASVR3MSCKASGYTFTSYVMHWVKQKPGQGLEWTG YVNPFNDGTKYNEMFKGKATI^ SLTSEDSAWYCARQAWGYPWGQGTT.ATVSA SEQ ID NO:449 Cell-targeting KEIGLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 11 LMIDSGIGDNLFAVDILGFDFTLGRFNA1LRLIVERNN LYVTGFVNRTNNVFYRF’ADFSHVI'FPGFFAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSLTQSVARAMLRIW"JA7TAEALRFRQIQRGFRTFL DDLSGASYVM1AEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRTSFGSINA1LGSVALTLNSHHHASAVAAEF PKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRAT1SC RATES VEYYGTSLVQWYQQKPGQPPKLL1YAASSV DSGWARFSGSGSGTDFSIYIHPVEEDDTAMYFCQQS RRVPYTFGGGTKLEIKGGGGSGGGGSGGGGSGGGG SGGGGSEVQLQQSGPELVKPGASY'KMSCKASGYTF TSYYAIHWYTxQKPGQGLEWGYYAIPFNDGTKAPxE MFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCA RQAWGYPWGQGTLVTVSA SEQ ID NO:450 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 12 LMIDSGIGDNLFAVDILGFDH’LGRFNNLRLIVERNN LYY'TGFVNRTNEiVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQLNRHSLTTSYLDLMSHS GTSLTQSVARAMT..RFVTVTAEALRFRQIQRGFRTTL DDT...SGASY\-A!TAEDVDLTLNVv־GRT...SSVLPD¥HGQ DSVRVGRISFGSLNAILGSVAUI.NSHHHASAVAAEF PKPSTPPGSSGGAPEIX'I.TQSPATI.SLSPGERATIvSCR ASQSVSSYI.،AWYQQKPGQ/\PRT..LIYDASNRATGIPA RFSGSGSGTDFn.TISST..EPEDFA\Y^CQQRSNWPTF GQGTKA'ETKGGGGSGGGGSGGGGSGGGGSGGGGS QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTY'AIS WVRQ/YPGQGLEWMGGIffIFGKM־IY־AQKI7QGRVTI TADESTSTAYMELSSLRSEDTAVYFCARKF'HFVSGS PFGMDVWGQGI’I'VI'VSS SEQ ID NO:451 Cell-targeting KEFrLJ.)FSTAKT¥VDST...NVTRSATGTPLQTISSGGTSL molecule 13 LYnDSGIGDNIEAVDILGFDFTL,GRF?GSlT..RlJVERNN [YVTGFVNRTNNYTYRFADFSHVTFPGTTAYTLSA DSSY^TTL,QRVAGISRTGMQINRHST..TTSYG..DIA/ISHS GTST..TQSVARA_MI,RFVTY7TAEAI..RFRQIQRGFRTTI, DDLSGASYAA4TAEDWT..n,NWGRLSS\7I.PDYT-IGQ DSVR.VGRTSFGSINAII.GSVAIJL.NSHHHASAVAAEF PKPS’FPPGSSGGM-’QVQLVQSGAiEVKKI-’GSSVKVSC RI'SGD'IFS'IYAISWVRQAJ-'GQGLEWMGGIIPIFGKA HYAQKF’QGRVnTADESTSTAYMELSSLRSEDTAVY FCARKIY-IFVSGSPFGAIDVWGQGT ’I'V’FVSSGGGGSG GGGSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGE RATLSCRASQSVSSY'LAWYQQKPGQAPRl.LIYDASWO 2016/196344 PCT/US2016/034778 400 NRATGIPARF'SGSGSGTDFI'LTISSLEPEDFAVYYCQ QRSNWPTFGQGTKVEIK SEQ ID NO:452 KEFTLDFSTAKTYVDSLNV1RSA1GTPLQTISSGGTSL Cell-targeting molecule 14 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRIT'v'ERNN IFYA'TGFYYJRTSnxWY'RFADFSHATFPGTTAAWL.SA DSSYTTI,QRVAGISRTGMQINRHSLTTSYI,DLMSHS GTSLTQSVARAMJIFVTVTAEALRFRQIQRGFRTTL DDI,SGASArVMTAED\a>LII..NAVGRI.,SS\l..PDAl-IGQ DSWVGRISFGSINAIL.GSVAIJI.NSFIHHASAVAAEF PKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRAriSC KASQSVDFDGDSYMNWYQQKPGQPPKVL1YAASN LESGIPARFSGSGSGTDFTLMHPVEEEDAAPYYCQQ SNEDPWTFGGGTKLE1KGGGGSQIQLQQSGPEVVKP GAS VKISCKASG Y'Il'I'D Y YITW VKQKPGQGLE WIG WIYPGSGNTKYNEKFKGKATLTVDTSSSTAF'MQLSS LTSEDTAVYFCANYGNYWFAYWGQGTQVTVSA SEQ ID NO:453 Cell-targeting KEFn.DFSTAKTYWSLNAlRSATGTPLQTISSGGTSL molecule 15 LMTDSGIGDMJEAATJUXiFDFTIXirtFA^ iA \ :Gi VXk : NTVi-A R:; \i): SHVi :;FG 11 AXELS X DSSY^TTL,QRVAGISRTGMQINRHST..TTSYG..DIA/ISHS GTSLTQSVARAMLRFVTVrAEALRERQIQRGFR.TTL DDLSGASYVA1TAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTU'C KASEDTYNRLTWYQQKPGKAPKLLISGATSLETGVP SRFSGSGSGrDFTFTISSLQPEDIATYYCQQYWSNPY TFGQGTKVETKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRG GSTDYNAAFMSRLNTFKDNSKNQVSLRLSSVTAAD TA VYYCAKSMITTGFVMDSWGQGSLVTVS S SEQ ID NO:454 KEFTLDFSTAKTYVDSLNVIRSAIG’TPLQTISSGGTSL Cell-targeting molecule 16 LMIDSGIGDNLFAVDILGFDFTLGRFNFILRLIVERMN LYVTGFVNRTNNVFYRF’ADFSHVI'FPGTT'AVTLSA DSSY1TLQRVAGISRTGMQINRIISLITSYLDLMSI-IS GTSLIQSVARAR/n.RFVTVTzAEzALPJ־RQIQRGFRTFL DDLSGASYVM’TAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRTSFGSINA1LGSVALTLNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC RASQDVN1AVAWYQQKPGKAPKLLIYSASFLYSGV PSRFSGSRSGTDFTI...TISST..QPEDFATYYCQQHYTTP PTFCiQGTKY'EIKGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGT.A;QPGGST..R.LSCAASGFN1KDTY1 HVv'VRQAPGKGLEWVARIYPTNGYTRYADSVKGRF nSADTSKNTAYLQMNSLRAEDTAVYY'CSRWGGD GFYAMDYWGQGTT.AWVSS SEQ ID NO:455 Cell-targeting KEFTLDFSTAKIYVDSLNV1R.SA1GTPLQTISSGGTSL molecule 17 LMIDSGIGDNLFAVDIEGFDFTLGRFNNLRLIVERNN LYVPGFVNR'PNAVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINPFiSLTTSYTDLMSHS GTSLTQSVARAMLRFVTVIAEALRF'RQIQRGFRITL DDLSGASYVNfTAEDVDLTIuNWGRLSSXTuPDYHGQ DSVRVGRISFGSLNAILGSVAUI.NSHHHASAVAAEF PKPSTPPGSSGGAPDIVMTQAAPSIPVTPGESVSISCR SSKSLLNSNGNTY1,YWFI.QRPGQSPQLLIYRMSNLA SGVPDRFSGSGSGTAFTI,RTSR\rEAEDVGVYWCMQWO 2016/196344 PCT/US2016/034778 401 HLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQL QQSGPELIKPGASVKAISCKASGYTFTSYVRRIWVKQ KPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDK SSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVFD YWGQGTTLTVSS SEQ ID NO:456 Cell-targeting KEFU.DFSTAKTYVDSLNVIRSAIGm.QTISSGGTSL molecule 18 IARDSGIGDNI,FAVDn..GFDFII,GRFNFn,RIJVERNN LYA'TGFXYJRTWWYWADFSHWFPGTTAWL.SA DSSYTTI,QR.VAGISRTGMQINRHSLTTSYI,DLMSHS GTSLrQSVARARILRFVTVTAEALFJRQIQRGFRTFL DDLSGASYVMTAEDVDL'ILNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMl^lTSSLSASLGDRVTISC PASQDISNYLAWYQQKI-’DGTVKLLIYYTSILHSGVP SRf'SGSGSGTDYSLTlSNLEQEDFATY'FCQQGNTLP WTFGCGTKLEIKGSTSGSGKPGSGEGSEVQLVESGG GLVKPGGSLKLSCAASGFAFSIYDMSWVRQIPEKC LEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLY LQMSSLKSEDTAMYYCARHSGYGTHWGVLFAYW GQGTLVTVSA SEQ ID NO:457 Cell-targeting KEFTLDFSTAKTYVDSLNV1RSAIGTPLQTISSGGTSL molecule 19 LM1D SGIGDNLF A VDILGFDFTLGRFNNLRL1VERNN LYVrGFVNRIMNVFYRI^zXDFSHVTFPGTTAVTLSzX DSSYTTLQRVAGISRTGMQIMRHSLTTSY'LDLMSHS GTSLTQSVARAMLREVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGR1SFGSINAILGSVAL1LNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC KASEDTYNRLTWYQQKPGKAPKLLISGATSLETGVP SRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPY TFGQGTKVE1KGSTSGSGKPGSGEGSTK.(.}QVQLQES GPGT..VRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGR GLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQ VSI.RLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSI.A׳rT\׳rSS SEQ ID NO:458 Cell-targeting KEF'rEDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL molecule 20 LMTDSGIGDNLFAVDILGFDFTLGPI-'NNLRLTVERNN EYVTGFVNRTNNYrFYRFADFSHVI'FPGr’IAV׳rLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTST..TQS V AR AMLRFVTVT AE ALRFRQIQR GFRTTL DDLSGASYVMTAEDY,DT..TLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVAIJLNSHHHASAVAAEF PKPSTPPGSSGGAPDIELTQSPSSFSVSLGDRYGTTCK ASEDIYNRLAWYQQKPGNAPRLLISGATSLETGV'PS RFSGSGSGKDYTT...SITST...QTEDVATY׳rYCQQYVvSTPT FGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSL VQPSQRLSTTCTVSGFSLTSYGVHW\,RQSPGKGLEW I.,G\1WGGSIT>YNAAFMSRLSITKDNSKSQVFFKM NSLQADDTATATCAKUJTTGYAMrnAVGQGTTVTV ss SEQ ID NO:459 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 21 LMIDSGIGDNLFAVDILGFVFTLGRFNNLRIAVTRNN LYVYGF VNRTNFi VF YRF ADFSH VTFPGTT AVTLS A DSSYTTLQRVAGISRTGMQLNRHSLTTSYLDLMSHS GTSLTQSVARAM..RFVTVTAEALRFRQIQRGFRTn..c9f:on a i Oas Q O' w WO 2016/196344 PCT/US2016/034778 402 DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGR1SFGSINAILGSVAL1LNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC KASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVP SRFSGSGSGrDFTFTISSLQPEDlATYYCQQYWSNPY TFGQGTKVETKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRG GSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TA WYCAKSM1TTGF W D S Vv GQGSL VTVS S SEQ ID NO:460 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Cell-targeting molecule 22 LMIDSGIGDNLFAVDILGFVFTLGRFNA1LRLIVERF1N LYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRI1SLITSYLDLMSHS GTSLIQSVARAR DDLSGASYVMTAEDVDL'ILNWGRLSSVLPDYHGQ DSVRVGRTSFGSINA1LGSVALTLNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC RASQGISS WL A WYQQKPEKAPKSLIYAAS SLQSG VP SRFSGSGSGTDF'TLTTSSLQPEDFAIYYCQQYNSYPY TFGQGTKLETKGGGGSQVQLVQSGAEVKKPGASVK VSCKASGYTFTSYDVHWVRQAPGQREEWMGWT..H ADTGrTKFSQKFQGRVTITRDTSASTAYMELSSLRSE DTAV'YYCARERIQLWFDYWGQGTIA'TVSS SEQ ID NO:461Cell-targeting KEF1LDFSTAKTYVDSLNV1RSAIGTPLQTISSGGTSL molecule 23 LMIDSCd'GDNLFAVDILGFVFTLGRFNNLRL LYVFGFVNR'FN־NVFYRFADFSHVTFPGTTAVTLSA DSSYTFLQRVAGISRl'GMQINRHSLTTSYLDLMSHS GTSLTQSVARAMLPJ'VTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAMSVALILNSHHHASAVAAEF PKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSC AASGFTFSDSWIHWVRQAPGKGLEWY'AWISPYGGS TYYADSVKGRFTISADTSKNTAYEQMNSLRAEDTA VYYCARRHWPGGFDYVv'GQGTEVTVSSGGGGSDlQ MTQSPSSLSASVGDRVnTCRASQDVSTAVA^QQ KPGKAPKT.IAYSASFLYSGWSRFSGSGSGTDFTLTTS SLQPEDFATYYCQQY1A1-IPATFGQGIKVEIK Cell-targeting KEFTLDFSTAKTYVDSLNVTRSATGTPLQT1SSGGTSL molecule 24 LMTDSGIGDNLFAVGILGFDFTLGPJ-'NNLRLTVERNN I.YVTGFyNRTNNYTYRFADFSHVTFPGTTAVTLSA DSSYTTLQRX'AGISRTGMQINRHSLTTSYI.DI.MSHS GTST..TQS V AR AMLRFVTVT AE ALRFRQIQR GFRTTL DDLSGASYVMTAEDVT)T..TLNWGRLSSVLPDYHGQ DSVRVGRTSFGSINAIEGSVAULNSHHHASAVAAEF PKPSTPPGSSGGAPDIQN4TQSPSSLSASVGDRVTITC K.ASEDIYNRLTWYrQQKPGKAPKLLISGATSLETGW SRFSGSGSGTDFTFTISSI.QPEDIATYATQQYWSNP’Y TFGQGTKVEIKGGGGSQVQLQESGPGL^7RPSQTI,SL TCTVSGFST.YSYGYTIWVRQPPGRGLEWTGYAIWG GSrDYNAAFMSPd.NnrKDNSKNQVSLRLSSVT/AAD TAVYYCAKSMITTOF VMD S WGQGSLVTVS S Cell-targeting KEFTLDFSTAKTYVDSLNVTRSAKjTPLQTISSGGTSL molecule 25 LMIDSGIGDNLFAVGILGFDFTLGRFNNLRIJVTFRNN L Y\׳׳TGF VNRTNA VF YRF ADFSH VTFPGTT AVTLS A DSSYTTI,QP..VAGISRTGMQIMtHSLTTSYT..DL.MSHSWO 2016/196344 PCT/US2016/034778 403 GTSLTQSVARAMLRFVTVGAEALRFRQIQRGFR1TI, DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGR1SFGSINAILGSVAL1LNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC RASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVP SRFSGSGSGIDFITIISSLQPEDFATYYCQQYNSYPY TFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVK VSCKASGV1TTSYDVHWVRQAPGQRLEWMGWLH ADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSE DTAVYYCARERIQLWTDYWGQGTLVTVSS SEQ ID NO:464 Cell-targeting KEIGLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 26 LMIDSGIGDNLFAVGILGFDFTLGRFNAILRLIVERNN LYVTGFVNRTNNVFYRFADFSHVIFPGTTAVILSA DSSYTTLQRVAGISRTGMQINRI-ISLITSYLDLMSHS GTSL’rQSVARAMLRFVTVTzAEzALFJ'RQIQRGFRTTL DDLSGASYVMIAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRTSFGSINA1LGSVALTLNSHHHASAVAAEF PKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSC aasgftfsdswthwvrqapgkglewvawispyggs TY'YADSVKGRFTISADTSKNTAYLQMNSLRAEDTA VYYCARRHVvPGGFDYWGQGTLVTVSSGGGGSDlQ MTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQ K.PGKAPKLLIYSASFIYSGVPSRFSGSGSGTDFTL..T1S SLQPEDFATYYCQQYI.YHPATFGQGTKVEIK SEQ ID NO:465 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 27 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN lyvpgfvnrtnavfyrfadfshvtfpgttavtlsa DSSYTFLQRVAGISRTGMQINRHSL1TSYLDLMSHS ATSLTQSVARAMLREVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSLNAILGSVAUI.NSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRX'TITC KASEDIYNRLTWYQQkPGKAPKIUSGATSLETGVT SRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSYPY IFGQGTK.\?ETKGGGGSQVQLQESGPGIA7RPSQII,SL TCTVSGFSLTSYrGVI-IWVRQPPGRGT.,EVtaG\?MWRG gstdynaafmsri,nitk.dnsknqvslrt.,ssvtaa.d TA VYYCAKSMITTGFVMD S WGQGSIAG'VS S SEQ ID NO:466 Cell-targeting KEF'ILDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL molecule 28 LAnDSGIGDNI,FAVDILGFDFTL,GRF?GSlT..RlJVERNN [.YVTGFVNRTNNYTYRFADFSHVTFPGTTAYTLSA DSSYTTLQRX'AGISRTGMQINRHSLTTSYI.DI.MSHS ATST..TQS V AR AMLRFVTVT AE ALRFRQIQR GFRTTL DDLSGRSYVMTAEDVDLTT..NWGRI,SSVT.PDYHGQ DSVRVGRISFGSINAILGSVAIJLNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSST..SASVGDRVTITC K.ASEDIYNRLTWYrQQKPGkAPKLLISGATSLETGW SRFSGSGSGTDFTFTISST.QPEDIATAATQQYAVSNP’Y TFGQGTKVEIK.GSTSGSGK.PGSGEGSTKGQVQLQES GPGLVRPSQILSLTCTVSGFSLTSYGVHWVRQPPGR GLEWIGVMWRGGSTDYNAA}־'MSRENITKDNSK1S؛Q VSLRLSSVTzAADrAVYYCzAKSAnTIGFVWSWGQ G SL VIV S SA SEQ ID NO:467 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 29 LMIDSGIGDNI.FAYDIT..GFDFTL.GRFNNI.RIJYTRKS1WO 2016/196344 PCT/US2016/034778 404 LYYG’GFVNRTFfNVFYRIGWFSHVTFPGTTzWTLSzA DSSYTTLQRVAGISRTGMQIFTRHSLTTSYLDLMSHS ATSLTQSVARAMLRFVTVrAEALRFM)IQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGR1SFGSINAILGSVAL1LNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC RASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGV PSRFSGSRSGTDFTUHSSLQPEDFAIYYCQQHYTTP PTFGQGTKVETKRTGSTSGSGKPGSGEGSEVQLVES GGGLVQPGGSLRLSCAASGFNIR7DTY1HWVRQAPG KGLFAVVARIYPTMPETRYADSVKGRFTISADTSKN TAYI.QMNSLRAEDTAVYYCSRWGGDGFYAMDW GQGTLVTVSS SEQ ID NO:468 Cell-targeting KE1GLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 30 LMIDSGIGDNLFAVDILGFDFTLGRFYNLRLIVERNN LYVTGFVNPJl'NNVFYRFADFSHVlFPGTTAVrLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS ATSLTQSVARAK4LRFVTV7TAEALRFRQIQRGFRTFL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVAIALNSHHHASAVAAEF PKPSTPPGSSGGAPDIELTQSPSSFSVSLGDR\GTTCK ASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPS RFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPT FGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSL VQPSQRLSTTCTVSGFSIJSYGVHWVRQSPGKGLEW LGMWGGSIDYNAAFMSRLSITKDNSKSQVFFKM NSLQADDTATATCAKHATTGYAMTnAVGQGTTVTV SS SEQ ID NO:469 Cell-targeting KEFTLDFSTAK'I'YVDSLNVIRSAIGTPLQTISSGGTSL molecule 31 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRI.I'v'ERNN LYY'TGFVNRTNMVFYRFADFSHVTFPGTTAVTLSA D S S YTTLQRVAGISRT GMQINRH SLTTSYLDLM SH S ATSLTQSVARAMT..RFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG(^ DSWVGRISFGSINAIL.GSVAIJI.NST-IHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC RA.SQDVNTAVAWYQQKPGKAPKLLTYSASFI..YSGV PSRFSGSRSGTDF׳n.TTSSLQPEDFATYYCQQEA؟TP PTFGQGTKVEHCGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGLVQPGGSLRLSCAASGFN1KDTYI HWVRQAPGKGLEWVARIYPTNGYFRYADSVKGRF IISADTSKNIAYLQMNSLRAEDTAVYYCSRWGGD GFYAMD Y WGQGTLVI'V S S SEQ ID NO:470 Cell-targeting KEFrL..DFSTAKTYVDST..NVTRSATGTPLQTISSGGTSL molecule 32 LYnDSGIGDNIPAVDILGFDFTL,GRF?GSlT..RlJVERNN :A \ tGL\A>GYX\lA RF \D: SHS'IFF(. :1 AX': LS X DSSYTTL.QRVAGISRTGMQINRHST..TTSYI..DIA/ISHS ATST..TQSVARA_MI,RFVTA7TAEAI.RFRQIQRGFRTTI, DDLSGRSYVA'ITAED\T)L.TT..NWGRIASM.PDA1-IGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAAEF’ PKPSTPPGSSGGAPDIVMTQzAAPSIPVTPGESVSISCR SSKSLLNSNGNTYLYWFLQRPGQSPQLLIYRMSNLP^ SGVPDRI^SGSGSGT^'ILRISRVEAEDVGVYYCMQ 1ILEYPFTFGAGTKLELKGSTSGSGKPGSGEG SEVQL QQSGPELIKPGASVKMSCKASGYTFTSYVAH-IWVKQr- 7־.
Q o w WO 2016/196344 PCT/US2016/034778 405 KPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDK SSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVFD YWGQGTTLTVSS SEQ ID NO:471Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 33 LMIDSGIGDNI..FAYDn..GFDFTL.GRFNNI,RIJATRKY LYV'TGFYAJRTWWYWADFSI-IWFPGTTAWL.SA DSSYTTLQRVAGISRTGMQEFfRHSIATSYtoLMSHS ATSIAQSVARAAnAtFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTT.NWGRLSSX'I.PDYHGQ DSVRVGRISFGSINAILGSVALILNSMHHASAVAAEF PKPSTPPGSSGGAPDIQMl^lTSSLSASLGDRVTISC PASQDISNYLAWYQQKPDGTVKLLIYYISILHSGVP SRf'SGSGSGTDYSLTlSNLEQEDFATYFCOQGNILP WTFGCGTKLEIKGSTSGSGKPGSGEGSEVQLVESGG GLVKPGGSLKLSCAASGFM׳'SrYDMSWVRQTPEKC LEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLY LOMSSLKSEDTAMYYCARHSGYGTHWGVLFAYW GQGTLVTVSA SEQ ID NO:472Cell-targeting KEFTLDFSTAKTY\Y)SLNVIRSATGTPLQTISSGGTST..
I..MTDSGIGDMFAWILGFDFTT.X؛RFNNT..P,I..TWRNN molecule 34 LYVGGFVNRINNVFYRIGWFSHVTFPGTTAVTLS/X DSSYTTLQRVAGISRTGMQIFTRHSLTTSYLDLMSHS ATSLTQSVARAMLRFVTVrAEALRFPXlIQRGFPATL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVAL1LNSHHHASAVAAEF PKPSrPPGSSGGAPDIVLTQSPASLAVSLGQRATTSC KASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASN LESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQ SNEDPWTFGGGTKLETKGGGGSQIQLQQSGPEVVKP GASVKISCKASGYTFTDYYITWW.QKPGQGLEWIG W1YPGSGNTKYNEKFKGKATLTVTDTSSSTAFMQLSS LTSEDTAVYFCANYGNYWFAYWGQGTQVTVSA SEQ ID NO:473 Cell-targeting KEFTLDFSTzXia'YVDSLNVIRSAIGTPLQTISSGGTSL molecule 3 5 LMIDSGIGDNLFAVDILGFDFILGRFNGLRLIVERNN LYVTGFVNRTNNVFYRF’ADFSHVI'FPGTIAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS ATSLTQSVARAK4LRFVTV7TAEALRFRQIQRGFRTrL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRTSFGSINA1LGSVALTLNSHHHASAVAAEF PRPPSTPPGSSGGAPQITIKESGPGILQPSQnAETCSF SGFSLTTYGIGVGWTRQPPGKGLEWI,THIWWNDNK YYNTAERSRLTTSKDSSNNQVUXIANVDTADTATY YCEYGYTYVv־GQGTEVTVSAGGGGSDVVMTQTPT..S I..PVSLGDQASISCRSSQSI.LYSNGNTYLHWYEQKPG QSPKLLTYKI.SNRFSGVPDRFSGSGSGTDFTLXISRV EAEDT..GWFCSQSTHWWTFGGGTKLETK Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL AMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN molecule 36 LYVPGFVNR'PNAVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINPFiSLTTSYLDLMSHS ATSLTQSVARAMT..RFVTVTAEAERFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGCj DSVRVGRISFGSLNAILGSVAUI.NSHHHASAVAAEF PKPSTPPGSSGGAPDIMAQSPASLAVSLGQRATTSC RATESXrEYYrGTSLVQWYQQKPGQPPKLLTYAASSVWO 2016/196344 PCT/US2016/034778 406 DSGVPARFSGSGSGTDFSLTHIPVEEDDIzXMYFCQQS rrvpytfgggtkleikggggsevqlqqsgpelvkpg ASVKMSCKASGYTFTSYVNRIWVKQKPGQGLEWIG YVNPFWGTKYNEYRKGKATLTSDKSSSTAYMELS SLTSEDSAVYYCARQAWGYPWGQGTLVTVSA SEQ ID NO:475 Cell-targeting KEFU.DFSTAKTYVDSLNVIRSAIGm.QTISSGGTSL molecule 37 LMIDSGIGDNI,FAVGn..GFWn,GRFNEn,RIJVERNN ؛ V\A GFVYR ؛ AYA ؛ V:>؛ ADPGA ؛'FFG ؛ ؛'A\ : PSA DSSY"TTI,QR.VAGISRTGMQINRHSLTTSYI,DLMSHS GTSLTQSVARAMLRFVTVTAEA^^ DDLSGASYVMTAEDVDL'ILNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAAEF PliPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC KASED1YNRLTWYQQKPGKAPKLLISGATSLETGVP SRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPY rFGQGTKVElKGGGGSQVQLQESGPGLVRPSQrLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWTGVMWRG GSTDYNAAFMSRLNITKDNSKNQVSLRASSVTAAD tavyycaksmtttgfvmdswgqgslvfvss SEQ ID NO:476 Cell-targeting KEFTLDFSTAKTYVT>SLNVTRSATGTPLQTISSGGTSL molecule 38 LM1D SGIGDNLF A VGILGFVFTLGRFNNLRL1VERNN LYAGGFVNRrFTNVFYRIAXDFSHVTFPGTTAVTLSzA DSSYTTLQRVAGISRTGMQINRHSLTTSY'LDLMSHS GTSLTQSVARAMLRFVTVIAEALRF^ DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC RASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVP SRFSGSGSGIDFTL'nSSLQPEDFATYYCQQYNSYPY TFGQGTKLEIKGGGGSQVQIArQSGAEVKKPGASVK VSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLH ADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSE DTAVYYCARERIQLVvFDYWGQGTLVTVSS SEQ ID NO:477 Cell-targeting KEF'ILDFS'IAKTYVDSLNVIRSAIG'IPLQTISSGGTSL LMIDSGIGDNLFAVGILGF’VFILGRFN^LRLIVERRN molecule 39 LYVTGFVNRTNNVFYRFADFSHVI'FPGr ’IAV'FLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSL'rQSVARAN'rLRFVTVTAEALRFRQIQRGFRTFL DDLSGASYVM1AEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVA.LILNSHHHASAVAAEF PKPSTPPGSSGGAPEVQIAT3SGGGLVQPGGSLRLSC AASGFTFSDSWTHWV'RQAPGKGLEWVAWISPYCKjS TY'YADSVKGRFTISADTSKNTAYLQMNSLRAEDTA VYYCARRHVvPGGFDYWGQGTLVTVSSGGGGSDlQ MTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQ KPGKAPKLLIYSASFI,YSGWSRFSGSGSGTDFTI.,TIS ST..QPEDFATArYCQQYI..YI-IPATFGQGTKAT,IK SEQ ID NO:478 Cell-targeting AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 40 LMIDSG1GDNLFAVDILGFDFTLGRFNNLRL1VERNN Lyvfgfvnr׳fn־nvfyrfadfshvtfpgttavtlsa DSSYTTLQRVAGISRTGMQLNRHSLTTSYLDLMSHS ATSLTQSVARAMT...RFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG(^ DSVRVGRISFGSLNAILGSVAUI.NSHHHASAVAAEF PKPSTPPGS SGGAPDIQMTQSPS SLS A SVGDRVTITCWO 2016/196344 PCT/US2016/034778 407 KASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVP SRFSGSGSGTDFlTlisSLQPEDIATYYCQQY'WSNPY TFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRG GSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TA VY YCAKSMHTGF VMD S WGOGSLVTVS S SEQ ID NO:479 Cell-targeting AEFU.DFSTAKTYVDSLNVIRSAIGm.QTISSGGTSL molecule 41 LMIDSGIGDNI^FAVDn..GFDFII^GRFNFn>RIJVERNN ITYV'TGFXTJRTWWWFADFSI-IWFPGTTAWL.SA DSSY1TLQRVAGISRTGMQINRIISLITSYLDLMSI-IS ATSLIQSVARARlLRFVTVTzXEzXLRl'RQIQRGFRTFL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC RASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGV PSRFSGSRSGTDFrLTlSSLQPEDFATYYCQQHYTTP PTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSL RLSCAASGFNIKD'TYIHWVRQAPGKGLEWVARIYP TNGYTRYADSVKGRFTISADTSKN'TAYLQMNSLRA edtavyycsrwggdgfyamdywgqgtlvtvssa SEQ ID NO:480 Cell-targeting KEFTLDFSTAKTYVDSLNV1RSAIGTPLQTISSGGTSL molecule 42 LM1D SGIGDNLF AVDILGFDFTLGRFNNLRL1VERNN LYYTGFVNRTPfNVFYRFzWFSHVTFPGTTzWTLSG DSSYTTLQRVAGISRTGMQINRHSLTTSYADLMSHS GTSLTQSVARAMLRJVTVTAEALRFRQIQRGFRGIL gdvftrsyvmtaedvdltlnwgrlssvlpdyhgq DSVRVGRISFGSINAILGSVALILNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC KASEDTYNRLTWYQQKPGKAPKLLISGATSLETGVP SRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSGPY TFCKJGTKVETKGSTSGSGKPGSGEGSTKilQVQLQES GPGLVRPSQTI.SLTCTVSGFSLTSYGVHWVRQPPGR GLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQ VSI.ALSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSI.A׳rT\׳rSS SEQ ID NO:481 Cell-targeting KEFTLDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSE molecule 43 LMTDSGIGDNLFAVDILGFDFTLGRI-'NNLRLTVERNN LYVTGFVNRTNNVFYRFADFSHVIFPGr’IAV'FLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS ATST..TQS V AR AMLRFVTVT AE ALRFRQIQR GFRTTL DDLSGASYVMTAEDY,DT..TLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVAIJLNSHHHASAVAAEF PKPSTPPGSSGGAPDIQN4TQSPSSLSASVGDR.VTITC KASEDIYNRI.TWYQQKPGKAPKLLISGATSLETGVP SRFSGSGSGTDFTFflSSI,QPEDIATYYCQQYWSNPY tfgqgtkytjkgstsgsgkpgsgegstkgqvqlqes GPGLVRPSQTL.SL.TCTVSGFSI.TSYGXTIWVRQPPGP..
GLE.WIGVAIWRGGSTDYrNAAFMSRT..NITKDNSKNQ VSLRLSSVTAADTAA^YYCAKSMITTGFVMDSWGQ GSLVPVSS SEQ ID NO:482 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 44 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRIAVT’RNN L YVYGF VNRTNFi VF YRF ADFSH VTFPGTT AVTLS A DSSYTTLQRVAGISRTGMQLNRHSLTTSYLDLMSHS ATSLTQSVARAMl..RFVTVTAEAI,RFRQIQRGFRTn.,WO 2016/196344 PCT/US2016/034778 408 DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC KASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVP SRFSGSGSGrDFTFTISSLQPEDIATYYCQQYWSNPY TFGQGTKVETKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRG GSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TA WYCAKSM1TTGF WD S Vv GQGSL VTVS S SEQ ID NO:483 Cell-targeting KEIGLDFSTAKTYVDSL/NVIRSAIGTPLQTISSGGT ’SL molecule 45 LMIDSGIGDNLFAVDILGFDFTLGRFYNLRLIVERF1N LYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYrTLQRVAGISRTGMQINRIISLITSYLDLMSHS ATSLIQSVARARlLRIWTVTzXEzXLPJ'RQIQRGFRTFL DDLSGASYVMTAEDVDLTLNWGRLSSVTPDYHGQ DSVRVGRTSFGSINA1LGSVALTLNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC RASQDVN1AVAWYQQKPGKAPKLLIYSASFLYSGV PSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTP PTFCiQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGTATIPGGSLRLSCAASGFNIKDTYI HVvVRQAPGKGLEWVARJYPTNGYTRYADSVKGRF TISADTSKNTAYLQMNSLRAEDTAVYY'CSRWGGD GFYAMDYWGQGTLVTVSS SEQ ID NO:484 Cell-targeting AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 46 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN LYYrFGFVNRTN־NVFYRFADFSHVTFPGTTAVTLSA DSSYTFLQRVAGISRTGMQINRHSLTTSYLDLMSHS ATSLTQSVARAMLRFVTVTAEALRRRQIQRGERTTL DDLSGASYVMTAEDVDLTI.NWGRLSSXT.PDYHGQ DSVRVGRISFGSLNAILGSVAUI.NSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRX'TITC KASEDIYNRLTWYQQkPGKAPKIUSGATSLETGVT SRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSYPY TFGQGTK.\?ETKGGGGSQVQLQE.SGPGIA7RPSQTI,SL TCTVSGFSLTSYrGVTIWVRQPPGRGT.,EVtaG\?MWRG GSTDYrNAAFMSRI.NITKDNSKNQVSLRT..SSWAAD TA VYYCAKSMITTGFVMI) SWGQGSLVTVS S SEQ ID NO:485 Cell-targeting KEFTLDFSTAKTYVDSLNV1RSA1GTPLQT1SSGGTSL molecule 47 LAnDSGIGDFII,FAVDILGFDFTL,GRF?GSlT..RLIVERNN [.YVTGFVNRTNNYTYRFADFSHVTFPGTTAYTLSA DSSYTTLQRX'AGISRTGMQINRHSLTTSYI.DI.MSHS ATST..TQS V AR AMLRFVTVT AE ALRFRQIQR GFRTTL DDLSGASYVMTAEDYT)T..TLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVAIJLNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSST..SASVGDRVTITC K.
SRFSGSGSGTDFTFTISST.QPEDIATAATQQYWSNP’Y TFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQn,SL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRG GSrDYNAAFMSPd.NnrKDNSKNQVSLRLSSVT/¥AD TA VYYCzXKSMl'IT GF VMD S WGOGSLVTVS S SEQ ID NO:486 Cell-targeting KEFILDFSTAK'n'VDSLNVTRSAIGTPLQTISSGGTSL molecule 48 LMIDSGIGDNLFAVDJLGFDFTLGRFNNLRIAVTFRNN LYA'TGFATJRTWWYRFADFSHWFPGTTAWL.SAWO 2016/196344 PCT/US2016/034778 409 DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS ATSLTQSVARAMLRFVTXTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC RASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGV PSRFSGSRSGTDFTUHSSLQPEDFA'IYYCQOHYTTP PTFGQGTKVETKGGGGSEVQLVESGGGLVQPGGSL RLSCAASGFNIKDTY'THWV'RQAPGKGLEWVARTYP TNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRA EDTA WYCSR WGGDGF YAMD Y WGQGTT... VTVS S A SEQ ID NO:487 Cell-targeting KEFILDFSTAKIY VDSLNV1RSA1GTPLQIISSGGTSL molecule 49 LMIDSGIGDNLFAVDILGFDFTLGRFYNLRLIVERNN LYVTGFVNRTNNVFYRFADFSHVrFPGTTAVI’LSA DSSY1TLQRVAGISRTGMQINRIISLITSYLDLMSI-IS ATSLTQSVARAK4LRFVTV7TAEALRFRQIQRGFRTFL DDLSGASYVMTAEDVDL1LNWGRLSSVLPDYHGQ DSVRVGRTSFGSINA1LGSVALTLNSHHHASAVAAEF PKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISC KASQSVDFrXIDSYMFAVYQQKPGQPPKVLlYAASN LESGIPARFSGSGSGTDFTLMHPVTEEDAATYYCQQ SNEDPWTFGGGTKTJF1KGGGGSQIQLQQSGPEVVKP GASVKISCKASGYTFTDYYTTWVKQKPGQGI.EWIG WIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSS LTSEDTAVYFCANYGNYWFA^YVGQGTQY^SA SEQ ID NO:488 Cell-targeting KEFILDFSTAKTYVDSLNVIRSATGTPLQTISSGGTSL molecule 50 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN lyvfgfvnrtn־nvfyrfadfshvtfpgttavtlsa DSSYTTLQRVAGISRTGMQLNPPiSLTTSYLDLMSHS ATSLTQSVARAMLRFVTV'TAEAI.RFRQIQRGFRTTL DDLSGASYVMTAEDVDLTI.NWGRLSS\,I.PDYHGQ DSVRVGRISFGSLNAILGSVAUI.NSHHHASAVAAEF PKPSTPPGSSGGAPDIVMTQAAPSIPVTPGESVSISCR SSKSLLNSNGNTY1,YWFI.QRPGQSPQLLIYRMSNLA SGVPDRFSGSGSGTAFTJ.,RTSR\rEAEDVGV¥YTMQ I־H7E'\7PFTFGAGTKLF.LKGSTSGSGKPGSGEGSEVQL QQSGPEL.IKPGAS\7KMSCKASGA؟FTSYVA׳n-IWWQ KPGQGLEWTGYINP’YFlDGTK’fNEKFKGKATLTSDK SSSTAYMELSSLTSEDSAY'Y'YCARGTYYYGSRVFD YWGQGITLTVSS SEQ ID NO:489 Cell-targeting QVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVH molecule 51 WVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATL TADKSSSTV'YMQLSSLTSEDSAWYCARSYYYGSS YVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQl VLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQ KPGSSPKPWTYATSNT..ASGVTARFSGSGSGTSYSLTT SRVEAEDAATYArCQQWISNPPTFGAGTKI,ELKEFPK PSTPPGSSGGAPKEFII.DFSTAKTYATtSLNMRSAIGT PT..QTISSGGTSI.LMIDSGIGDNI..FAYDIT..GFDFTL.GRF N^LRLIVERNNLYVrGFVRRTNNVFYRF'ADFSHVTF PGrTAVlLSADSSYTTLQRVAGISRIGMQlNRllSLT TSYLDLMSHSATSLTQSVARAMLRIAG'VTzAEzALRf־•' RQIQRGFRIl'LDDLSGASYVMIAEDVDLTLNWGRL SSVLPDYHGQDSVRVGRISFGS1NAILGSVALILNSH FII-lASAVAzAWO 2016/196344 PCT/US2016/034778 410 SEQ ID NO:490 Cell-targeting KEE1LDFSI(AKTYVDSLNVIRSAIGTPLQTISSGGTSL LM1D SGIGDNLF A VDILGFDFTLGRFNNLRL1 VERNN molecule 52 LYVrGFVNRrFTNVFYRIGADFSHVTFPGTTAVTLSzA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS ATSLTQSVARAMLRJVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGR1SFGSINAILGSVAL1LNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQTTSSLSASLGDRVTISC RASQDISNYLAWYQQKPDGTVKLLIYYTSrLHSGVP SRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTT..P WTFGCGTKLEIKGSTSGSGKPGSGEGSEVQLVESGG GLVKPGGSLKLSCAASGFAFSiYDMSWVRQTPEK.C LEWVAYISSGGGTTYYPDIYKGRFTTSRDNAKNTLY LQMSSLKSEDTAMYYCARHSGYGTH.WGVI.FAYW GQGTLVTVSA SEQ ID NO:491 Cell-targeting KEFTLDFSTAKIY'VDSLNVIRSAIGTPLQTISSGGTSL LMTDSGIGDNLFAVDILGFDFTLGPTNNLRLIVERNN molecule 53 LYVTGFVNRTNNVFYRFADFSHVI'FPGF’IAV'rLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS ATST..TQS V AR AMLRFVTVT AE ALRFRQIQR GFRTTL DDLSGASYVMTAEDWT..TLNWGRLSSVLPDYHGQ DSVRVGRTSFGSINAILGSVAULNSHHHASAVAAEF PKPSTPPGSSGGAPDIQN4TQSPSSLSASVGDRVTITC KASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVP SRFSGSGSGTDFTFTTSSLQPEDIATYYCQQYWSFiPY TFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQES GPGIA^PSQTLSLTCTVSGFSLTSYGVHWVRQPPGR GLEWIG\7MWRGGSTDYNzAAFMSRLNITK.DNSKNQ VSLRLSSVTAADTAA^YYCAKSMITTGFVMDSWGQ GSLVTVsS SEQ ID NO:492 Cell-targeting KEFILDFSTAK'n'VDSLNVTRSAIGTPLQnSSGGTSL molecule 54 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRIAVTRNN LYVTGFVNRTNAiVFYRFADFSHVTFPGTTAVTLSA D S S YTTEQRVAGISRT GMQINRH SETTS YLDLM SH S ATSLTQSVARAYn..RFVTVTAEAIRFRQIQRGFRTTL, DDT..SGASYWTAEDW2LT1..NWGRT..SS\7I..PDAT-IGQ DSWVGRISFGSINAIL.GSVAUI.NSFIHHASAVAAEF PKPSTPPGSSGGAPDIELTQSPSSFSVST..GDRVTITCK ASEDIYARLAWYQQKPGNAPRLLISGATSLETGVPS RF'SGSGSGKDYTLSITSLQTEDVA'rYYCQQYWSTPT FGGG'IKLEIKGSTSGSGKPGSGEGSKVQLQESGPSL vqpsqrlsitctvsgfslisygvi-iwvrq'spgkglew LGVIWRGGSTDYAAAF'MSRLSITKDNSKSQVFFKM NSLQADDI/XIYFCzAKTLITrGYAMDYWGQGTrV'rV ss SEQ ID NO:493 Cell-targeting KEFTI.DFSTAKTYVDSLNATR.SAIGTPLQTISSGGTSL L.MTDSGIGD:FF.EA\T)ILGFDFTI..GRFNNT..P.IJWRNN molecule 55 :a \ Rr \D: SHS'1 TFCHI •w: LS X DSSY^TTL.QRVAGISRTGMQINRHST..TTSYT.DIA/ISHS AI'SLTQSV^AMLRI-VTVI'.AE.ALRFRQIQRGFRTTL DDLSGASYVAFI’AEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAAEF’ PKPSTPPGSSGGAPQITLKESGPGILQPSQTLSLTCSF SGFSLITYGIGVGWIRQPPGKGLEWLTIHWWNDNK YYNTALRSRLTISKDSSNNQVLLKIANVDTADI'AI'YWO 2016/196344 PCT/US2016/034778 411 Y CLYG YTY WGQGTL VT VSAGGGG SD VVMTQTPLS LPVSLGDQASISCRSSQSLLYSNGN'I'YLI-IWYLQKPG QSPKLLrYKLSNRFSGVPDPFSGSGSGTDFTLKISRV EAEDLGVYFCSQSTHVPWTFGGGTKLEIK SEQ ID NO:494 Cell-targeting KFFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 56 LMIDSGIGDNI.FAYDIT..GFDFTL.GRFNNI.RIJYTEKY ؛ V\G(>F\ \R : \\\؛ V:<: AD؛G؛؛\ ؛ LSA DSSYTTLQRVAGISRTGMQIMU4SIYTSYLDLMSHS ATSLTQSVARA^^..RFVTVTAEAI,RFRQIQRGFRT^..
DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSEIHHASAVAAEF PKFSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC KASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGV PSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTP pifgqgikveikggggsggggsggggsggggsggg GSEVQEVESGGGLVQPGGSLRASCAASGFNIKDTYI HWVRQAPGKGLEWVARIYPTNGY’IRYADSVKGRF T1SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGD GFYAMDYWGQGTLVTVSS SEQ ID NO:495 Cell-targeting KEFU.DFSTAKTYVDSLNYTR.SAIGTPLQTISSGGTSL molecule 57 LM1D SGIGDNLF AVDILGFDFTLGRFNNLRL1VERNN LYVrGFVNRrPTNVFYRIGADFSHVTFPGTTAVTLSzA DSSYTTLQRVAGISRTGMQIFTRHSLTTSYTDLMSHS ATSLTQSVARAMLRFVTVI'AEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDETLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAA'EF PKPSTPPGSSGGAPD1VLTQSPASLAVSLGQRATTSC RATESVEYYGTSLVQWYQQKPGQPPKLLTYAASSV DSGVPARFSGSGSGrDFSLTIHPVEEDDIAMYFCQQS RRVTYTFGGGTKLETKGGGGSEVQI.QQSGPELVKPG ASVKMSCKASGYTFTSYVMHWVKQKPGQGLEVvTG YY7NPFNDGTKYNEMFKGKATLTSDKSSSTAYMET...S SLTSEDSAYYYCARQAWGYPWGQGTIATVSA SEQ ID NO:496 Cell-targeting zXEFILDFSTAKIYVDSLNVlRSAlGTPLQIISSGGTSL LMIDSGIGDNLP'AVDILGFDFILGRFNGLRLIVERNN molecule 58 LYVTGFVNRTNN^/FYPJ־;ADFSHVI'FPGr’IAV׳rLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS ATSLrQSVARAMERFVTVTAEALPdvRQIQRGFRTrL DDLSGASYVMIAEDVDL1LNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVA.LILNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSST..SASVGDRVTITC KASEDIYNRI.TWYQQKPGKAPKLLISGATSLETGVP SRFSGSGSGTDFTFflSSI,QPEDIATYYCQQYWSNPY TTCiQGTKVElKGGGGSQVQLQESGPGLVRPSQTLvSL TCTVSGFSI.TSYGVHWVRQPPGRGLEWTGVMWRG GSTDY7NAAFMSRLNn׳KDNSKNQVSI.,RLSSVTAAD TAXY'YC A K SMITTGFX'MD S WGQGSL VTVS S SEQ ID NO:497 Cell-targeting AEFILDFSTAKTYVDSLNVIRSATGTPLQTISSGGTSL molecule 59 LMfDSGIGDNLFAVNLY'PMVATVGRFNNLRLTVERN NLYV'FGFVNRrNNVFYRl'-ADFSHVTFPGTTAVTLS ADSSYTTT..QRVAGTSRTGMQINRHSI.TTSYLDLMSH SATSLTQSVARAMI.RFVTVTAEAI,RFRQIQRGFRTT LDDLSGASYVMTAED\7DLn,NWGRLSSVT״PDYHG QDSVRVGRISFGSrNATLGSVAULNSHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITWO 2016/196344 PCT/US2016/034778 412 CKASEDIYNRLTWY'QQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNP YTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWR GGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSW^^ SEQ ID NO:498 Cell-targeting KFYILDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL molecule 60 LMIDSGIGDNITAVD\rRGIAPIEARFFINI،RT..I\T,RNN LYA'TGFYTJRTWWYWADFSHYT’FPGTTAWL.SA DSSY1TLQRVAGISRTGMQINRIISLTTSYLDLMSI-IS ATSLIQSVARARlLRIWTVTzAEzALPJ'RQIQRGFRTFL AALSGASYVMTAEDVDL'ILNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILGSVALILNSHHHASAVAAEF PI^STPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC KASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVP SRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPY ■IFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQFLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWTGVMWRG GSTDYNAAFMSRLNITKDNSKNQVSLPASSVTAAD TAV YYC AK SMITTGFVMD S W GQGSL VT VS S SEQ ID NO:499 Cell-targeting KEFTLDFSTAKIYVDSLNV1R.SAIGTPLQTISSGGTSL molecule 61 LM1D SGIGDNLF AVDILGFDFTLGRFNNLRLIVERNN LYYGGFVNRTFTNVFYRFzWFSHV^ DSSYTTLQRVAGISRTGMQINRHSLTTSY'LDLMSHS ATSLTQSVARAMLPJ-'VTVIAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGR1SFGSINAILGSVAL1LNSHHHARNLVPMV ATVASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLS ASVGDRVTITCKASEDIYNRLTWYQQkPGKAPKLLI SGATST...ETG\-TSRFSGSGSGTDFTFTTSSLQPEDIArn׳r YCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGP GIArRPSQTLSLTCTVSGFSI.TSYGVHWVRQPPGRGI, EWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVS LRI.SSX'TAADTAVYYCAKSMITTGFV'MDSWGQGSL VTVSS SEQ ID N0:500 Cell-targeting KEF'1־LDFSTAKTYVDSLNVTRSATGTPLQT1SSGGTSL molecule 62 LMTDSGSGDNLFAVDTLGFDF'ILGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHV’I'FPGr'IAV'rLS GDSSYlTLQRVAGlSRTGMQTNRHSLTrSYLDLMSH SGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTr LDDLSGRSYVMTAEDV'DLTL.NWGRLSSVIPDYHG QDSVRVGRTSFGSINAII.GSVALILNCHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTIT CKASEDIYNRLTWY'QQKPGkAPKIUSGATSLETGV PSRFSGSGSGTDFTFTISST..QPEDIATYYCQQYWSNP YTFGQGTKW.IKGGGGSQVQT..QESGPGLA7RPSQTT..S L.TCTVSGFSLTSYGVT-IWVRQPPGRGLE.WIGVF4W GGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAA'AA'YSAKSMITTGFVF/IDSWGQGST.ATVSS SEQ ID NO:501 Cell-targeting K'EFTLDFSTAKTYVDSLNVIRSAIG'rPLQTlSSGGTSL molecule 63 LMIDSGSGDNLFAVD'v'RGIAPEEGRFMNLRLTVERN GLYATGFVNRTMNVFYRFADFSHVTFPGTTAVTLS ADSSYTTT..QRVAGISRTGMQINRHSI.TTSYLDLMSH SATSLTQSVARAMI.RFVTVTAEAI.RFRQIQRGFRTT LDDLSGASTrVMTAEDWLTl.،NWGRLSSV[..PDYTIGWO 2016/196344 PCT/US2016/034778 413 QDSVRV GR1SFG SINAILGS VALILN SHHHASAVAAE FPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVT1T CKASED1YNRLTWYQQKPGKAPKLLISGATSLETGV PSRFSGSGSGTDFTFG1SSLQPEDIATYYCQQYWSNP YTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQE SGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPG RGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKN QVSLRLSSV"I'AADTAVYYCAKSMTTrGFVMDSWG QGSLVTVSSA SEQ ID NO:502 Cell-targeting zAEFTLDFSTzAia'YVDSLNVIRSAIGTPLQTISSGGTSL molecule 64 LMIDSGIGDNLFAVDILGFDFTLGRFNGLRLIVERNN EYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS zVrSL’rQSVARAMLRFVTVTzAEzALFJ'RQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRTSFGSINA1LGSVALTLNSHHHASAVAAEF PKPSTPPGSSGGAPDVQVTQSPSSLSASVGDRVTTTC RSSQSLANSYGNTFLSWYLHKPGKAPQLLIYGISNR FSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCLQG THQPYTFGQGIXVETKGGGGSEVQLVQSGAEVKKP GASVKVSCKASGYRFTNYWIHWVRQAPGQGLEWI GGINPGNNYATY'RRKFQGRVTMTADTSTSTVYMEL SSLRSEDTAVYYCTREGYGNYGAAVFAYWGQGTLy TVS SA SEQ ID NO:503 Cell-targeting AEFTLDFSTAK1YVDSLNVIRSAIGTPLQT1SSGGTSL molecule 65 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRL1VEP.NN LYYG'GFVNRTNRVFYRFADFSHVTFPGTTAVTLSA DSSYTFLQRVAGISRIGMQINRHSLITSYLDLMSHS ATSLTQSVARAMLRFVTVIAEALRF'RQIQRGFRrf'L DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG(^ DSVRVGRISFGSLNAILGSVAUI.NSHHHASAVAAEF PKPSTPPGSSGGAPDIMYQSPASLAVSLGQRATTSC KASQSVDFDGDSYMNWYQQKPGQPPKVLTYAASN LESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQ SNEDPWTFGGGIKLETKGGGGSQIQLQQSGPEWTCP GAS\rKISCKASGYTFTDA,YITWVk.QKPGQGLEWIG WIYTGSGNTKYrNE,KFKGKATT..TWTSSSTAFMQT.RS LTSEDT A AGTCANYGNYAVT AYAVGQGTQWVS A SEQ ID NO:504 Cell-targeting KEFTLDFSTAKTYVDSLNVTRSATGTPLQTISSGGTSL molecule 66 LYnDSGIGDNI,FAVDILGFDFTL,GRF?TNT..RlJVERNN [YVTGFVNRTNNWYRFADFSHVTFPGTTAYTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYI,DI,MSHS ATST..TQS V AR AMLRFVTVT AE ALRFRQIQR GFRTTL DDLSGRSYVMTAEDVDLTT..RWGRLSSYT..PDYHGQ DSVRVGRISFGSINAILGSVAIALNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSST..SASVGDRVTITC K.ASEDIYNRLTWArQQKPGkAPKLLISGATSLETGW SRFSGSGSGIDFTFTISSI.QPEDIATY^CQQYAVSRW TFGQGTKVEIKGGGGSQVQLQESGPGL^7RPSQTI,SL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRG GSrDYNAAFMSRLMITKDNSKNQVSLRLSSVT/AAD TA VY YC AKSM1TTGF’ VMD S WGOGSLVTVS SA SEQ ID NO:505 Cell-targeting KEFILDFSTAK'n'VDSLNVTRSAIGTPLQnSSGGTSL molecule 67 LMIDSGIGDNLFAVDVRGIAPIEAP.FNYLRT...IVERNR LYA'TGFAYJRTWWYRFADFSHWFPGTTAWL.SAWO 2016/196344 PCT/US2016/034778 414WO 2016/196344 PCT/US2016/034778 415 LYVrGFVNRIFTNVFYRIGWFSHVTFPGTTAVTLSzX DSSYTTLQRVAGISRTGMQLYRHSLTTSYLDLMSHS ATSLTQSVARAMLRFVTVTAEALRFRCMQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGR1SFGSINAILGSVAL1LNSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC KASEIWNRLTWYQQKPGKAPKLLISGATSLETGVP SRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPY TFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEW1GVN4WRG GSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TA VYYCAKSMITTGFVMDSWGQGSLVTVS SA SEQIDNO:510 Cell-targeting KEFILDFSTAKIYVDSLNV1RSA1GTPLQIISSGGTSL molecule 73 LMIDSGIGDNLFAVD1LGFDFTLGRFYNLRLIVERNN LYVTGFVNRTNNVFYRFADFSHVTFPGTTAVI’LSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS ATSLTQSVARAK^LRFVTV'TAEALRFRQIQRGFRTFL DDLSGASYVMIAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRTSFGSINA1LGSVALTLNSHHHASAVAAEF PKPSTPPGSSGGAPDIQN4TQSPSSLSASVGDRVTITC KASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVP SRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPY TFGQGTKVTJKGGGGSQVQIX^ESGPGLVRPSQTLSL TCTVSGFSI.TSYGVHWVRQPPGRGLEWTGVMWRG GSTDWA.AFMSRT.,NITKDNSKNQVST..RI.,SSVTAAD TAVYYC A K SMiTTGFVMD S WGQGSL VTVS S A SEQ1DNO:511 Cell-targeting AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 74 LMIDSG1GDNLFAVDILGFDFTLGRFNNLRL1VERNN Lyvfgfvnr׳fn־nvfyrfadfshvtepgttavtlsa DSSYTTLQRVAGISRTGMQLNRHSLTTSYLDLMSHS ATSLTQSVARAMT...RFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHG(^ DSVRVGRISFGSLNAILGSVAUI.NSHHHASAVAAEF PKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC RA.SQDVNTAVAWYQQKPGKAPKLLTYSASFI..YSGV PSRFSGSRSGTDFTI.TTSSLQPEDFATYYCQQG^fTTP PTFGQGTK.VEIKGGGGSEVQLV’ESGGGLVQPGGSL RT.. SC A AS GFNIKDTYTH W W Q APGK GLE W VARTAT ■Ilx؛GYTRY/ADSVKGRl-׳nSADTSKN؛I'AYLQMNSLRA ED'EAVYYCSRWGGDGFYAMDYWGQG’I'LVTVSSA SEQIDNO:512 Cell-targeting AEFTI...DFSTAKTYVDST...NVTRSATGTPLQTISSGGTST.. molecule 75 [A!TDSGIGDNIEAVDILGFDFTI.GRFlxNI...RLIVERKiN [YVTGFVNRTNNWYRFADFSHVTFPGTTA'vTLSA DSSYTTLQRX'AGISRTGMQINRHSLTTSYI.DI.MSHS ATST..TQS V AR AMLRFVTVT AE ALRFRQIQR GFRTTL DDLSGRSXrX;MTAEDXTJLTl.,NWGRLSSVTPDYTIG<) DSVR.VGRTSFGSINAII.GSVAI.JL.NSHHHASAVAAEF PKPSIPPGSSGGAPAIQMSQSPA.SLSASVGETVTITC RASGNIYNYI..AYX^rQQKQGKSPTlI..T.,VYDAKTI,ADG VPSRF'SGSGSGTQYSLKISSLQTEDSGNYYCQEFWS LPFTFG SGTKLE1KGSTSG SGKPG SGEG Sl'KGE VQLQ QSGAEL VRPG׳XL VfiLSCKTSGFNIKOYFLH WVRQR PDQGLEWIGWINPDNGNTVYDPKF'QGTASLTADTS SNTVYLQLSGLTSEDTAVYFCTRRDYTYEKAALDY ago■ הר \ :WO 2016/196344 PCT/US2016/034778 416 SEQIDNO:513 Cell-targeting AEFTLDFSTAKTYVDSLNVORSAIGTPLQTISSGGTSL LMID SGIGDNLF A VDILGFDFTLGRFNNLRL1VERNN molecule 76 LYVrGFVNRIFTNVFYRIGWFSHVTFPGTTAVTLSzX DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS ATSLTQSVARAMLRFVTVIAEALRF'RQIQRGFRITL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGR1SFGSINAILGSVAL1LNSHHHASAVAAEF PKPSTPPGSSGGAPDIQLTQSPLSLPVTLGQPASTSCR SSQSLVHRNGNTYLHWFQQRPGQSPRLLIYTVSNRF SGWDRFSGSGSGTOFnXISRVTAEDVGVYFCSQSS HVVPTFGAGTRLFJKGSTSGSGKPGSGEGSTKGQVQ LQQSGSELKKPGASVKVSCKASGYTTTNYGVNWK QAPGQGI.QWY1GWINPNTGEPTFDDDFKGRFAFSLD TSVSTAYI..QISSLKADDTAVYFCSRSRGKNEAWFA YW G QGTL.WVS S417 285032/2

Claims (12)

1. A Shiga toxin effector polypeptide comprising an amino acid sequence having at least 90% identity to amino acids 1 to 251 of SEQ ID NO: 1, wherein the amino acid sequence comprises: i) a heterologous, CD8+ T-cell epitope embedded at amino acid positions 53 – 66 of SEQ ID NO: 1 and having the amino acid substitutions: V54I, R55L, I57F, P59F, E60T, and E61L, and ii) the amino acid substitutions: S45I, G110A, R188A, C242S, R248A and R251A; and wherein the amino acid sequence comprises an asparagine at the amino acid residue corresponding to position 75 of SEQ ID NO: 1, a tyrosine at the amino acid residue corresponding to position 77 of SEQ ID NO: 1, a tyrosine at the amino acid residue corresponding to position 114 of SEQ ID NO: 1, a glutamate at the amino acid residue corresponding to position 167 of SEQ ID NO: 1, an arginine at the amino acid residue corresponding to position 170 of SEQ ID NO: 1, an arginine at the amino acid residue corresponding to position 176 of SEQ ID NO: 1, and a tryptophan at the amino acid residue corresponding to position 203 of SEQ ID NO: 1.
2. The Shiga toxin effector polypeptide according to claim 1, wherein the Shiga toxin effector polypeptide comprises an amino acid sequence having at least 95% sequence identity to amino acids 1 to 251 of SEQ ID NO: 1.
3. A cell-targeting molecule comprising i) a binding region capable of specifically binding an extracellular target biomolecule physically coupled to the cellular surface of a cell, and ii) the Shiga toxin effector polypeptide of claim 1 or claim 2.
4. The cell-targeting molecule of claim 3, wherein the binding region is fused to the carboxy terminus of the Shiga toxin effector polypeptide to form a single, continuous polypeptide. 418 285032/2
5. The cell-targeting molecule of claims 3 or 4, wherein the binding region comprises an immunoglobulin-type binding region.
6. The cell-targeting molecule of claim 5, wherein the immunoglobulin-type binding region comprises a polypeptide selected from: single-domain antibody fragment, single-chain variable fragment, antibody variable fragment, complementary determining region 3 fragment, constrained FR3-CDR3-FR4 polypeptide, Fd fragment, antigen-binding fragment, fibronectin- derived 10th fibronectin type III domain, tenascin type III domain, ankyrin repeat motif domain, low-density-lipoprotein-receptor-derived A-domain, lipocalin, Kunitz domain, Protein-A-derived Z domain, gamma-B crystallin-derived domain, ubiquitin-derived domain, Sac7d-derived polypeptide, Fyn-derived SH2 domain, miniprotein, C-type lectin-like domain scaffold, a heavy- chain antibody domain derived from a camelid V H fragment, heavy-chain antibody domain H derived from cartilaginous fish, immunoglobulin new antigen receptor (IgNAR), V fragment, NAR multimerizing scFv fragment, bivalent minibody, bispecific tandem scFv, bispecific tandem VHH, or bispecific minibody.
7. The cell-targeting molecule of claim 6, wherein the multimerizing scFv fragment comprises a diabody, a triabody, or a tetrabody
8. The cell-targeting molecule of any one of claims 3-7, which comprises the linker peptide shown in SEQ ID NO: 540-543 or 544-550, or 553-559.
9. The cell-targeting molecule of any one of claims 3-7, which comprises the linker peptide shown in SEQ ID NO: 540-543 or 553-557.
10. A pharmaceutical composition comprising the cell-targeting molecule of any one of claims 3-9 and a pharmaceutically acceptable excipient or carrier.
11. A polynucleotide encoding the cell-targeting molecule of any one of claims 3-9, or a complement thereof. 419 285032/2 12. An expression vector comprising the polynucleotide of claim 11. 13. A host cell comprising the polynucleotide of claim 11 or the expression vector of claim
12. For the Applicants REINHOLD COHN AND PARTNERS By:
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